Pyrrolobenzodiazepines

ABSTRACT

Compounds of the formulae Ia and Ib: 
                 
 
wherein:
         A is CH 2 , or a single bond;   R 2  is selected from: R, OH, OR, CO 2 H, CO 2 R, COH, COR, SO 2 R, CN;   R 6 , R 7  and R 9  are independently selected from H, R, OH, OR, halo, amino, NHR, nitro, Me 3 Sn;   and R 8  is selected from H, R, OH, OR, halo, amino, NHR, nitro, Me 3 Sn, where R is as defined above, or the compound is a dimer with each monomer being the same or different and being of formula Ia or Ib, where the R 8  groups of the monomers form together a bridge having the formula —X—R′—X— linking the monomers, where R′ is an alkylene chain containing from 3 to 12 carbon atoms, which chain may be interrupted by one or more hetero-atoms and/or aromatic rings and may contain one or more carbon-carbon double or triple bonds, and each X is independently selected from O, S, or N;   except that in a compound of formula Ia when A is a single bond, then R 2  is not CH═CH(CONH 2 ) or CH═CH(CONMe 2 ). Other related compounds are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of prior U.S. patentapplication Ser. No. 09/763,767, filed on Feb. 26, 2001 which is afiling under 35 U.S.C. 371 based upon International Application No.PCT/GB99/02838, filed on Aug. 27, 1999, which claims priority to GreatBritain Application No. 9818733.9, filed on Aug. 27, 1998 and GreatBritain Application No. 9901929.1, filed on Jan. 28, 1999.

BACKGROUND TO THE INVENTION

Some pyrrolobenzodiazepines (PBDS) have the ability to recognise andbond to specific sequences of DNA; the preferred sequence is PuGPu. Thefirst PBD antitumour antibiotic, anthramycin, was discovered in 1965(Leimgruber et al., 1965 J. Am. Chem. Soc., 87, 5793-5795; Leimgruber etal., 1965 J. Am. Chem. Soc., 87, 5791-5793). Since then, a number ofnaturally occurring PBDs have been reported, and over 10 syntheticroutes have been developed to a variety of analogues (Thurston et al.,1994 Chem. Rev. 1994, 433-465). Family members include abbeymycin(Hochlowski et al., 1987 J. Antibiotics, 40, 145-148), chicamycin(Konishi et al., 1984 J. Antibiotics, 37, 200-206), DC-81 (JapanesePatent 58-180 487; Thurston et al., 1990, Chem. Brit., 26, 767-772; Boseet al., 1992 Tetrahedron, 48, 751-758), mazethramycin (Kuminoto et al.,1980 J. Antibiotics, 33, 665-667), neothramycins A and B (Takeuchi etal., 1976 J. Antibiotics, 29, 93-96), porothramycin (Tsunakawa et al.,1988 J. Antibiotics, 41, 1366-1373), prothracarcin (Shimizu et al, 1982J. Antibiotics, 29, 2492-2503; Langley and Thurston, 1987 J. Org. Chem.,52, 91-97), sibanomicin (DC-102) (Hara et al., 1988 J. Antibiotics, 41,702-704; Itoh et al., 1988 J. Antibiotics, 41, 1281-1284), sibiromycin(Leber et al., 1988 J. Am. Chem. Soc., 110, 2992-2993) and tomamycin(Arima et al., 1972 J. Antibiotics, 25, 437-444). PBDs are of thegeneral structure:

They differ in the number, type and position of substituents, in boththeir aromatic A rings and pyrrolo C rings, and in the degree ofsaturation of the C ring. In the B-ring there is either an imine (N═C),a carbinolamine (NH—CH(OH)), or a carbinolamine methyl ether(NH—CH(OMe)) at the N10-C11 position which is the electrophilic centreresponsible for alkylating DNA. All of the known natural products havean (S)-configuration at the chiral C11a position which provides themwith a right-handed twist when viewed from the C ring towards the Aring. This gives them the appropriate three-dimensional shape forisohelicity with the minor groove of B-form DNA, leading to a snug fitat the binding site (Kohn, 1975 In Antibiotics III. Springer-Verlag, NewYork, pp. 3-11; Hurley and Needham-VanDevanter, 1986 Acc. Chem. Res.,19, 230-237). Their ability to form an adduct in the minor groove,enables them to interfere with DNA processing, hence their use asantitumour agents.

DISCLOSURE OF THE INVENTION

A first aspect of the present invention is a compound with the formulaIa or Ib:

wherein:

-   -   A is CH₂, or a single bond;    -   R₂ is selected from: R, OH, OR, CO₂H, CO₂R, COH, COR, SO₂R, CN;    -   R₆, R₇ and R₉ are independently selected from H, R, OH, OR,        halo, amino, NHR, nitro, Me₃Sn;    -   where R is a lower alkyl group having 1 to 10 carbon atoms, or        an aralkyl group (i.e. an alkyl group with one or more aryl        substituents), preferably of up to 12 carbon atoms, whereof the        alkyl group optionally contains one or more carbon-carbon double        or triple bonds, which may form part of a conjugated system, or        an aryl group, preferably of up to 12 carbon atoms; and is        optionally substituted by one or more halo, hydroxy, amino, or        nitro groups, and optionally containing one or more hetero atoms        which may form part of, or be, a functional group; or R₇ and R₈        together from a group —O—(CH₂)_(p)—O—, where p is 1 or 2;    -   and R₈ is selected from H, R, OH, OR, halo, amino, NHR, nitro,        Me₃Sn, where R is as defined above, or the compound is a dimer        with each monomer being the same or different and being of        formula Ia or Ib, where the R₈ groups of the monomers form        together a bridge having the formula —X—R′—X— linking the        monomers, where R′ is an alkylene chain containing from 3 to 12        carbon atoms, which chain may be interrupted by one or more        hetero-atoms and/or aromatic rings, e.g. benzene or pyridine,        and may contain one or more carbon-carbon double or triple        bonds, and each X is independently selected from O, S, or N;        except that in a compound of formula Ia when A is a single bond,        then R₂ is not CH═CH(CONH₂) or CH═CH(CONMe₂)

If A is a single bond then R₂ is bonded directly to the C-ring of thePBD.

If R is an aryl group, and contains a hetero atom, then R is aheterocyclic group. If R is an alkyl chain, and contains a hetero atom,the hetero atom may be located anywhere in the alkyl chain, e.g.—O—C₂H₅, —CH₂—S—CH₃, or may form part of or be a functional group e.g.carbonyl, hydroxy.

It is preferred that in a compound of formula Ia when A is a singlebond, then R₂ is not CH═CR^(A)R^(B), where R^(A) and R^(B) areindependently selected from H, R^(C), COR^(C), CONH₂, CONHR^(C),CONR^(C) ₂, cyano or phosphonate, where R^(C) is an unsubstituted alkylgroup having 1 to 4 carbon atoms.

R is preferably selected from a lower alkyl group having 1 to 10 carbonatoms, or an aralkyl group, preferably of up to 12 carbon atoms, or anaryl group, preferably of up to 12 carbon atoms, optionally substitutedby one or more halo, hydroxy, amino, or nitro groups. It is morepreferred that R is selected from a lower alkyl group having 1 to 10carbon atoms optionally substituted by one or more halo, hydroxy, amino,or nitro groups. It is particularly preferred that R is an unsubstitutedstraight or branched chain alkyl, having 1 to 10, preferably 1 to 6, andmore preferably 1 to 4, carbon atoms, e.g. methyl, ethyl, n-propyl,n-butyl or t-butyl.

Alternatively, R₆, R₇, R₉ and, unless the compound is a dimer, R₈ maypreferably be independently selected from R groups with the followingstructural characteristics:

-   (i) an optionally substituted phenyl group;-   (ii) an optionally substituted ethenyl group;-   (iii) an ethenyl group conjugated to an electron sink.

The term ‘electron sink’ means a moiety covalently attached to acompound which is capable of reducing electron density in other parts ofthe compound. Examples of electron sinks include cyano, carbonyl andester groups.

It may be preferred that A is CH₂ and/or that R₂ is CO₂H, CO₂R, CH₂OH,or CH₂OR. It may be further preferred that R₂ is CO₂Me, CO₂ ^(t)Bu,CH₂OH, or CH₂OAc.

R₆, R₇, and R₉, unless the compound is a dimer, R₈ are preferablyselected from H and OR, and more particularly H, OMe and OCH₂Ph. It isfurther preferred that R. and, unless the compound is a dimer, R₈ areOR, more preferably OMe or OCH₂Ph, and that R₆ and R₉ are H.

If A is a single bond, then R₂ is preferably an aryl group, eg Ph,p-MeO—Ph, or an alkyl or alkaryl group which contains at least onedouble bond which forms part of a conjugated system with the double bondof the C-ring, eg CH═CH₂, CH═CH—CH₃.

Compounds of the first aspect of the invention are preferably of formulaIa.

If the compound of formula Ia or Ib is a dimer, the dimer bridge may beof the formula —O—(CH₂)_(p)—O—, where p is from 1 to 12, more preferably3 to 9.

A second aspect of the present invention is a compound with the formulaII:

wherein:

-   -   R′₂ is selected from: O, CHR″₂, where R″₂ is selected from H, R,        CO₂R, COR, CHO, CO₂H, halo;    -   R₆, R₇ and R₉ are independently selected from H, R, OH, OR,        halo, amino, NHR, nitro, Me₃Sn;    -   where R is a lower alkyl group having 1 to 10 carbon atoms, or        an aralkyl group (i.e. an alkyl group with one or more aryl        substituents), preferably of up to 12 carbon atoms, whereof the        alkyl group optionally contains one or more carbon-carbon double        or triple bonds, which may form part of a conjugated system, or        an aryl group, preferably of up to 12 carbon atoms; and is        optionally substituted by one or more halo, hydroxy, amino, or        nitro groups, and optionally containing one or more hetero atoms        which may form part of, or be, a functional group;    -   and R₈ is selected from H, R, OH, OR, halo, amino, NHR, nitro,        Me₃Sn, where R is as defined above or the compound is a dimer        with each monomer being the same or different and being of        formula II, where the R₈ groups of the monomers form together a        bridge having the formula —X—R′—X— linking the monomers, where        R′ is an alkylene chain containing from 3 to 12 carbon atoms,        which chain may be interrupted by one or more hetero-atoms        and/or aromatic rings, e.g. benzene or pyridine, and may contain        one or more carbon-carbon double or triple bonds, and each X is        independently selected from O, S, or N; or R₇ and R₈ together        form a group —O—(CH₂)_(p)—O—, where p is 1 or 2;    -   except that:        -   (i) when R₁₂ is CH—Et, and R₆, R₈ and R, are H, R₇ is not            sibirosamine pyranoside; and        -   (ii) when R′₂ is CH—Me, and R₆ and R₉ are H, R₇ and R₈ are            not both H or both Ome, or OMe and OH respectively.

If R is an aryl group, and contains a hetero atom, then R is aheterocyclic group. If R is an alkyl chain, and contains a hetero atom,the hetero atom may be located anywhere in the alkyl chain, e.g.—O—C₂H₅, —CH₂—S—CH₃, or may form part of or be a functional group e.g.carbonyl, hydroxy.

R is preferably selected from a lower alkyl group having 1 to 10 carbonatoms, or an aralkyl group, preferably of up to 12 carbon atoms, or anaryl group, preferably of up to 12 carbon atoms, optionally substitutedby one or more halo, hydroxy, amino, or nitro groups. It is morepreferred that R is selected from a lower alkyl group having 1 to 10carbon atoms optionally substituted by one or more halo, hydroxy, amino,or nitro groups. It is particularly preferred that R is an unsubstitutedstraight or branched chain alkyl, having 1 to 10, preferably 1 to 6, andmore preferably 1 to 4, carbon atoms, e.g. methyl, ethyl, n-propyl,n-butyl or t-butyl.

Alternatively, R₆, R₇ and R₉ and, unless the compound is a dimer, R₈ maypreferably be independently selected from R groups with the followingstructural characteristics:

-   (i) an optionally substituted phenyl group;-   (ii) an optionally substituted ethenyl group;-   (iii) an ethenyl group conjugated to an electron sink.

R′₂ is preferably O, CH₂ or CHCH₃ and more preferably CH₂ or CHCH₃.

R₆, R₇, and R₉ and, unless the compound is a dimer, R₈ are preferablyselected from H and OR and a halogen atom, and more particularly H, OMeand OCH,Ph, and I. It is further preferred that R₇ and, unless thecompound is a dimer, R₈ are OR or a halogen atom, more preferably OMe,OCH₂Ph or I, and that R₆ and R₉ are H. Most preferably R₈ is BnO.

If the compound of formula II is a dimer, the dimer bridge may be of theformula —O—(CH₂)_(p)—O—, where p is from 1 to 12, more preferably 3 to9, and most preferably 3 to 5.

A third aspect of the present invention is a compound with the formulaIII:

wherein:

-   -   R₆, R₇ and R₉ are independently selected from H, R, OH, OR,        halo, amino, NHR, nitro, Me₃Sn;    -   where R is a lower alkyl group having 1 to 10 carbon atoms, or        an aralkyl group (i.e. an alkyl group with one or more aryl        substituents), preferably of up to 12 carbon atoms, whereof the        alkyl group optionally contains one or more carbon-carbon double        or triple bonds, which may form part of a conjugated system, or        an aryl group, preferably of up to 12 carbon atoms;    -   and is optionally substituted by one or more halo, hydroxy,        amino, or nitro groups, and optionally containing one or more        hetero atoms which may form part of, or be, a functional group;    -   and R₈ is selected from H, R, OH, OR, halo, amino, NHR, nitro,        Me₃Sn, where R is as defined above or the compound is a dimer        with each monomer being the same or different and being of        formula III, where the R₈ groups of the monomer form together a        bridge having the formula —X—R′—X— linking the monomers, where        R′ is an alkylene chain containing from 3 to 12 carbon atoms,        which chain may be interrupted by one or more hetero-atoms        and/or aromatic rings, e.g. benzene or pyridine, and may contain        one or more carbon-carbon double or triple bonds, and each X is        independently selected from O, S, or N; or R₇ and R₈ together        form a group —O—(CH₂)_(p)—O—, where p os 1 or 2;    -   wherein at least one of R₆, R₇, R₈ and R₉ are not H;    -   except that:        -   (i) when R₆ and R₉ are H, R₇ and R₈ are not both OMe, OMe            and OBn respectively, or OMe and OH respectively;        -   (ii) when R₆ and R₇ are H, R₈ and R₉ are not Me and OH            respectively;        -   (iii) when three of R₆, R₇, R₈ and R₉ are H, the other is            not Me;        -   (iv) when R₆, R₇, and R₈ are H, R, is not OMe;        -   (v) when R₆, R₈ and R₉ are H, R₇ is not OMe; and        -   (vi) when R₆, and R₉ are H and R₇ is OMe, the compound is            not a dimer.

If R is an aryl group, and contains a hetero atom, then R is aheterocyclic group. If R is an alkyl chain, and contains a hetero atom,the hetero atom may be located anywhere in the alkyl chain, e.g.—O—C₂H₅, —CH₂—S—CH₃, or may form part of or be a functional group e.g.carbonyl, hydroxy.

R is preferably selected from a lower alkyl group having 1 to 10 carbonatoms, or an aralkyl group, preferably of up to 12 carbon atoms, or anaryl group, preferably of up to 12 carbon atoms, optionally substitutedby one or more halo, hydroxy, amino, or nitro groups. It is morepreferred that R is selected from a lower alkyl group having 1 to 10carbon atoms optionally substituted by one or more halo, hydroxy, amino,or nitro groups. It is particularly preferred that R is an unsubstitutedstraight or branched chain alkyl, having 1 to 10, preferably 1 to 6, andmore preferably 1 to 4, carbon atoms, e.g. methyl, ethyl, n-propyl,n-butyl or t-butyl.

Alternatively, R₆, R₇ and R₉ and, unless the compound is a dimer, R₈,may preferably be independently selected from R groups with thefollowing structural characteristics:

-   (i) an optionally substituted phenyl group;-   (ii) an optionally substituted ethenyl group;-   (iii) an ethenyl group conjugated to an electron sink.

It is preferred that either:

-   (i) only one of R₆, R₇, R₈ and R₉ is H; or-   (ii) at least one of R₆, R₇, R₈, and R₉ is NH₂; or-   (iii) at least one of R₆, R₇, R₈ and R₉ is an aryl group, preferably    of up to 12 carbon atoms, which is optionally substituted by one or    more halo, hydroxy, amino, or nitro groups, and optionally contains    one or more hetero atoms which may form part of, or be, a functional    group.

If only one of R₆, R₇, R₈ and R₉, it is further preferred that theA-ring substituents (i.e. those of R₆, R₇, R₉ and, unless the compoundis a dimer, R₈ which are not H) are OR, and are more preferably selectedfrom OMe, and OBn.

If at least one of R₆, R₇, R₈ and R₉ is an aryl group, preferably of upto 12 carbon atoms, which is optionally substituted by one or more halo,hydroxy, amino, or nitro groups, and optionally contains one or morehetero atoms which may form part of, or be, a functional group, it isfurther preferred that at least one of R₆, R₇, R₈ and R₉, is a phenylgroup optionally substituted by one or more methoxy, ethoxy or nitrogroups, although the nitro groups are less preferred. More preferably,the aryl group is selected from: Ph and p-MeO—Ph.

If the compound of formula III is a dimer, the dimer bridge may be ofthe formula —O—(CH₂)_(p)—O—, where p is from 1 to 12, more preferably 3to 9. Also in this case, it is preferred that R₆ and R₉ are H, and R₇ isan alkoxy or aryloxy group.

A fourth aspect of the present invention provides a compound with theformula IV:

wherein:

-   -   R₆, R₇ and R₉ are independently selected from H, R, OH, OR,        halo, amino, NHR, nitro, Me₃Sn;    -   where R is a lower alkyl group having 1 to 10 carbon atoms, or        an aralkyl group (i.e. an alkyl group with one or more aryl        substituents), preferably of up to 12 carbon atoms, whereof the        alkyl group optionally contains one or more carbon-carbon double        or triple bonds, which may form part of a conjugated system, or        an aryl group, preferably of up to 12 carbon atoms;    -   and is optionally substituted by one or more halo, hydroxy,        amino, or nitro groups, and optionally containing one or more        hetero atoms which may form part of, or be, a functional group;    -   R₈′ and R₈″ are either independently selected from H, R or        together form a cyclic amine; and    -   n is from 1 to 7.

If R₈′ and R₈″ form a cyclic amine, then there is usually a single Natom in a ring which is otherwise carbocyclic and is preferably 5- or6-membered and may be saturated or unsaturated. The ring may be fused toanother ring system which may be aromatic, e.g. being a benzene ring.Thus for example the cyclic amine may be an indolyl or isoindolyl group.It is also possible that the cyclic amine contains one or more heteroatoms, in addition to N in the amine ring and/or in a fused ring and mayalso be substituted by one or more R groups.

If R is an aryl group, and contains a hetero atom, then R is aheterocyclic group. If R is an alkyl chain, and contains a hetero atom,the hetero atom may be located anywhere in the alkyl chain, e.g.—O—C₂H₅, —CH₂—S—CH₃, or may form part of or be a functional group e.g.carbonyl, hydroxy.

R is preferably selected from a lower alkyl group having 1 to 10 carbonatoms, or an aralkyl group, preferably of up to 12 carbon atoms, or anaryl group, preferably of up to 12 carbon atoms, optionally substitutedby one or more halo, hydroxy, amino, or nitro groups. It is morepreferred that R is selected from a lower alkyl group having 1 to 10carbon atoms optionally substituted by one or more halo, hydroxy, amino,or nitro groups. It is particularly preferred that R is an unsubstitutedstraight or branched chain alkyl, having 1 to 10, preferably 1 to 6, andmore preferably 1 to 4, carbon atoms, e.g. methyl, ethyl, n-propyl,n-butyl or t-butyl.

It may be preferred that one of R′₈ and R″₈ is a nitrogen protectinggroup, such as Fmoc.

R₇ is preferably an electron donating group, and is more preferably ofthe formula OR; particularly preferred are the groups OMe, OEt, and OBn.The term ‘electron donating group’ means a moiety covalently attached toa compound which is capable of increasing electron density in otherparts of the compound.

In addition R₆ and R₉ are more preferably selected from H and OR;particularly preferred are Ome, OEt and OBn.

Alternatively, R₆, R₇ and R₉ may preferably be independently selectedfrom R groups with the following structural characteristics:

-   (i) an optionally substituted phenyl group;-   (ii) an optionally substituted ethenyl group;-   (iii) an ethenyl group conjugated to an electron sink.

n is preferably 1 to 3, and more preferably 1.

A fifth aspect of the present invention is the use of a compound asdescribed in the first, second, third or fourth aspects of the inventionin a method of therapy. Conditions which may be treated includegene-based diseases, including, for example, neoplastic diseases andAlzheimer's Disease, and also bacterial, parasitic and viral infections.Any condition which may be treated by the regulation of gene expressionmay be treated using compounds of the invention. In accordance with thisaspect of the present invention, the compounds provided may beadministered to individuals. Administration is preferably in a“therapeutically effective amount”, this being sufficient to showbenefit to a patient. Such benefit may be at least amelioration of atleast one symptom. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofwhat is being treated. Prescription of treatment, e.g. decisions ondosage, is within the responsibility of general practitioners and othermedical doctors.

A compound may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated.

Pharmaceutical compositions according to the present invention, and foruse in accordance with the present invention, may comprise, in additionto the active ingredient, i.e. a compound of formula Ia, Ib, II, III orIV, a pharmaceutically acceptable excipient, carrier, buffer, stabiliseror other materials well known to those skilled in the art. Suchmaterials should be non-toxic and should not interfere with the efficacyof the active ingredient. The precise nature of the carrier or othermaterial will depend on the route of administration, which may be oral,or by injection, e.g. cutaneous, subcutaneous, or intravenous.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may comprise a solid carrier oran adjuvant. Liquid pharmaceutical compositions generally comprise aliquid carrier such as water, petroleum, animal or vegetable oils,mineral oil or synthetic oil. Physiological saline solution, dextrose orother saccharide solution or glycols such as ethylene glycol, propyleneglycol or polyethylene glycol may be included. A capsule may comprise asolid carrier such a gelatin.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.

A sixth aspect of the present invention is a pharmaceutical compositioncontaining a compound of any one of formulae Ia, Ib, II, III, or IV asdescribed above, and a pharmaceutically acceptable carrier or diluent.The preparation of pharmaceutical compositions is described in relationto the fifth aspect of the invention above.

A seventh aspect of the present invention provides the use of a compoundof any one of formulae Ia, Ib, II, III, or IV as described above toprepare a medicament for the treatment of a gene-based disease,preferably a proliferative disease. The compound of formula Ia, Ib, II,III, or IV may be provided together with a pharmaceutically acceptablecarrier or diluent. The compounds may be used for the selective killingof oxic and hypoxic tumour cells in methods for the treatment ofcancers, for example leukemias and particularly solid cancers includingcolon, CNS, renal, and lung tumours, including small cell lungcarcinoma, and melanomas. In particular, dimers of formula II may beused for the selective killing of lung, colon, and CNS tumours andmelanomas. The compounds of formula III and IV may be used selectivelyagainst melanomas.

A further aspect of the present invention provides the use of a compoundof any one of formulae Ia, Ib, II, III, or IV as described above toprepare a medicament for the treatment of a viral, parasitic orbacterial infection. The preparation of a medicament is described inrelation to the fifth aspect of the invention above.

In further aspects, the invention provides processes for preparingcompounds according to the first, second, third and fourth aspects ofthe present invention.

Aspects of the invention will now be further described with reference tothe accompanying drawings in which:

FIGS. 1 to 6 a/b are synthesis routes for compounds of formula Ia of thepresent invention;

FIGS. 7 to 14 are synthesis routes for compounds of formula II of thepresent invention;

FIGS. 15 to 24 are synthesis routes for compounds of formula III of thepresent invention;

FIG. 25 is a synthesis route for a compound of formula IV;

FIG. 26 is a synthesis of an intermediate in the preparation ofcompounds of formula IV of the present invention;

FIG. 27 is a synthesis routes for compounds of formula IV of the presentinvention; and

FIGS. 28 to 31 are graphs illustrating the cytotoxicity results ofexamples 5 to 8 respectively.

Preferred General Synthetic Strategies

A key step in a preferred route to compounds of formula Ia, Ib, II, IIIor IV is a cyclisation to produce the B-ring, involving generation of analdehyde (or functional equivalent thereof) at what will be the11-position, and attack thereon by the Pro-N10-nitrogen:

In this structure, no C-ring substitution or unsaturation is shown. R₈represents O(CH₂)_(n)CH₂COR′₈ in compounds of formula IV. R₁₀ is anitrogen protecting group, preferably with a carbamate functionalitybonded to the nitrogen of the PBD. The “masked aldehyde” —CPQ may be anacetal or thioacetal (possibly cyclic), in which case the cyclisationinvolves unmasking. Alternatively, the masked aldehyde may be analdehyde precursor, such as alcohol —CHOH, in which case the reactioninvolves oxidation, e.g. by means of TPAP or DMSO (Swern oxidation).

The masked aldehyde compound can be produced by condensing acorresponding 2-substituted pyrrolidine with a 2-nitrobenzoic acid:

The nitro group can then be reduced to —NH₂ and protected by reactionwith a suitable reagent, e.g. a chloroformate, which provides theremovable nitrogen protecting group in the synthesis route.

A process involving the oxidation-cyclization procedure is illustratedin scheme 1 (an alternative type of cyclisation will be described laterwith reference to scheme 2).

The imine/carbinolamine bond in the PBD (A) can be unprotected bystandard methods to yield the desired compound, e.g. if R₁₀ is Alloc,then the deprotection is carried out using palladium to remove the N10protecting group, followed by the elimination of water to give theimine.

Exposure of the alcohol (B) (in which the Pro-N10-nitrogen is generallyprotected as carbamate) to tetrapropylammonium perruthenate(TPAP)/N-methylmorpholine N-oxide (NMO) over A4 sieves results inoxidation accompanied by spontaneous B-ring closure to afford thedesired product. The TPAP/NMO oxidation procedure is found to beparticularly convenient for small scale reactions while the use ofDMSO-based oxidation methods, particularly Swern oxidation, provessuperior for larger scale work (e.g. >1 g).

The uncyclized alcohol (B) may be prepared by the reaction of a nitrogenprotection reagent of formula D, which is preferably a chloroformate oracid chloride, to a solution of the amino alcohol C, generally insolution, generally in the presence of a base such as pyridine(preferably 2 equivalents) at a moderate temperature (e.g. at 0° C.).Under these conditions little or no O-acylation is usually observed.

The key amino alcohol C may be prepared by reduction of thecorresponding nitro compound E, by choosing a method which will leavethe rest of the molecule intact. Treatment of E with tin (II) chloridein a suitable solvent, e.g. refluxing methanol, generally affords, afterthe removal of the tin salts, the desired product in high yield.

Exposure of E to hydrazine/Raney nickel avoids the production of tinsalts and may result in a higher yield of C, although this method isless compatible with the range of possible C and A-ring substituents.For instance, if there is C-ring unsaturation (either in the ringitself, or in R₂ or R₃), this technique may be unsuitable.

The nitro compound of formula E may be prepared by coupling theappropriate o-nitrobenzoyl chloride to a compound of formula F, e.g. inthe presence of K₂CO₃ at −25° C. under a N₂ atmosphere. Compounds offormula F can be readily prepared, for example by olefination of theketone derived from L-trans-hydroxy proline. The ketone intermediate canalso be exploited by conversion to the enol triflate for use inpalladium mediated coupling reactions.

The o-nitrobenzoyl chloride is synthesised from the o-nitrobenzoic acid(or alkyl ester after hydrolysis) of formula G, which itself is preparedfrom the vanillic acid (or alkyl ester) derivative H. Many of these arecommercially available and some are disclosed in Althuis, T. H. andHess, H. J., J. Medicinal Chem., 20(1), 146-266 (1977).Alternative Cyclisation (Scheme 2)

In scheme 1, the final or penultimate step was an oxidative cyclisation.An alternative, using thioacetal coupling, is shown in scheme 2.Mercury-mediated unmasking causes cyclisation to the protected PBDcompound (A).

The thioacetal compound may be prepared as shown in scheme 2: thethioacetal protected C-ring [prepared via a literature method: Langley,D. R. & Thurston, D. E., J. Organic Chemistry, 52, 91-97 (1987)] iscoupled to the o-nitrobenzoic acid (or alkyl ester after hydrolysis) (G)using a literature procedure. The resulting nitro compound cannot bereduced by hydrogenation, because of the thioacetal group, so thetin(II) chloride method is used to afford the amine. This is thenN-protected, e.g., by reaction with a chloroformate or acid chloride,such as 2,2,2-trichloroethylchloroformate.

Acetal-containing C-rings can be used as an alternative in this type ofroute with deprotection involving other methods, including the use ofacidic conditions.Dimer Synthesis (Scheme 3)

PBD dimers may be synthesized using the strategy developed for thesynthesis of the protected PBD monomers. The synthesis routesillustrated in scheme 3 show compounds when the dimer linkage is of theformula —O—(CH₂)_(n)—O—. The step of dimer formation is normally carriedout to form a bis(nitro acid) G′. This compound can then be treated ascompound G in either scheme 1 or scheme 2 above.

The bis(nitro acid) G′ may be obtained by nitrating (e.g. using 70%nitric acid) the bis(carboxylic acid). This can be synthesised byalkylation of two equivalents of the relevant benzoic acid with theappropriate diiodoalkane under basic conditions. Many benzoic acids arecommercially available and others can be synthesised by conventionalmethods. Alternatively, the relevant benzoic acid esters can be joinedtogether by a Mitsunobo etherification with an appropriate alkanediol,followed by nitration, and then hydrolysis (not illustrated).

An alternative synthesis of the bis(nitro acid) involves oxidation ofthe bis(nitro aldehyde), e.g. with potassium permanganate. This can beobtained in turn by direct nitration of the bis(aldehyde), e.g. with 70%HNO₃. Finally, the bis(aldehyde) can be obtained via the Mitsunobuetherification of two equivalents of the benzoic aldehyde with theappropriate alkanediol.

An alternative synthesis approach to those detailed above is to protectthe pro N10 position on the component which will form the A-ring, beforejoining the component which will form the C-ring.

Preferred Synthetic Strategies for Compounds of Formula Ia

The synthesis route of scheme 1 is generally applicable to compounds offormula Ia.

C2/C3-endo-unsaturated PBDs of formula Ia may be synthesised from theirN10-carbamate protected precursors. Typically, palladium catalysedremoval of an allyl carbamate may be used to generate the N10-C11 iminewithout affecting the key C2-unsaturation. For example, if the N10-C11imine/carbinolamine is protected by an Alloc group, theC2/C3-endo-unsaturation is maintained during the Alloc cleavagereaction.

The reduction of the nitro-compound E as shown in scheme 1 with tin (II)chloride in refluxing methanol leaves the C2/C3-unsaturation unaffected.The hydrazine/Raney nickel method would not be suitable due to thedouble bond.

The compound of formula F may be used in its TBDMS protected form, andtherefore a deprotection step has to be included to produce theamino-alcohol compound E.

The TBDMS ether, which is the product of the coupling of TBDMS protectedcompound with the appropriate o-nitrobenzoyl chloride, can be treatedwith AcOH:THF:H₂O (3:1:1). TBAF was found to be unsuitable for thistransformation due to the rapid degradation of reaction products.

A class of requisite C-ring providing compounds F can be obtained asshown in scheme 4.

Commercially available trans-4-hydroxy-L-proline F8 can be N-allocprotected to give the allyl carbamate F7 which can then be esterifiedusing standard conditions. Hydride reduction of the ester F6 furnishesthe diol F5. Selective TBDMS protection of the diol gives a silyl etherF4, which can then be oxidised, using either Swern or TPAP oxidation, toprovide the ketone F3.

The ketone F3 can then undergo a Wittig reaction to yield a mixture ofthe E/Z exo-esters F2 which can then be converted to the C2/C3-endocompound F1(Ia) upon treatment with excess sodium hydride.Palladium-mediated cleavage of the N-alloc protecting group (Dangles 0.;Guibé, F.; Balavoine, G.; Lavielle, S.; Marquet, A.; J. Org. Chem. 1987,52, 4984) yields the compound F(Ia).

Alternative Route to Compounds of Formula Ia

A more linear synthetic route to compound B of scheme 1 has beendeveloped which enables larger scale production of theC2/C3-endo-unsaturated PBDs, and is shown in scheme 5.

The silyl protecting group may be cleaved in good yield by treatingB1(Ia) with AcOH:THF:H₂O (3:1:1). The key C2/C3-endo-unsaturationpresent in B1(Ia) may be introduced directly by performing theHorner-Emmons reaction on a ketone of type B2. Unlike the previous route(Scheme 4), the addition of extra NaH to ensure double-bond migrationwas not necessary for this substrate. Swern oxidation of the secondaryalcohol B3 may be used to furnish the ketone B2. The carbamate protectedaniline B3 may be prepared from the nitro compound B5 in two steps.Firstly, the nitro group may be reduced to the aniline by employing theRaney nickel/hydrazine method because a compound of type B5 lacksC2-unsaturation. This method is more advantageous than the tin (II)chloride procedure since the product is easier to isolate. The anilineB4 may then be N-carbamate protected in high yield without significantcarbonate formation at the C2 oxygen.An amide of type B5 may be synthesised by coupling an acid chloride oftype G to the key amine KEC5 (Scheme 6).

Overall, this route has several advantages over the previous route whichresults in the larger scale production of the C2/C3-endo-unsaturatedPBDs. Firstly, catalytic hydrogenation of KEC4 allows large scalepreparation of key intermediate KEC5. Secondly, this more efficientnitro reduction step may be carried out on an intermediate devoid ofC2-unsaturation. Importantly, the double-bond migration observed duringthe Horner-Emmons reaction is spontaneous, so excess sodium hydride isnot necessary. This double-bond migration has also been observed byother workers (Leimgruber, W.; Batcho, A. D.; Czajkowski, R. C. J. Am.Chem. Soc. 1968, 90, 5641).

Parr-hydrogenation of KEC4, in order to cleave the Cbz protecting group,allowed the large scale synthesis of the key amino intermediate KEC5.The TBDMS ether KEC4 was prepared in an analogous fashion to thecorresponding Alloc protected intermediate F4 (Scheme 4). Selectivesilylation of the primary alcohol KEC3 was achieved using DBU as a silyltransfer agent. The diol KEC3 was obtained from hydride reduction ofester KEC2 which in turn was synthesised from carboxylic acid KEC1.N-Cbz protection of trans-4-hydroxy-L-proline (F4) was achieved byadopting a procedure reported in the literature (Bridges, R. J.;Stanley, M. S.; Anderson, M. W.; Cotman, C. W.; Chamberlain, R. A. J.Med. Chem. 1991, 34, 717).

Certain R₂ groups may require protection during the synthesis routes setout above, e.g. alcohols can be protected by using an acetate protectinggroup (see example 1(d))Further Alternative Route to Compound of Formula Ia

The following route is particularly suited to a compound of formula Iawhere A is a single bond, and R₂ is an allyl group or contains a doublebond which is conjugated to that in the C-ring. However, elements of thesynthesis, eg the SEM protection, may be useful in a route to othercompounds.

The target PBDs were prepared by reduction of the SEM protected dilactam(Q) with sodium tetraborohydride followed by treatment with silica gel.The sodium tetraborohydride, initially, converts the dilactam into aprotected carbinolamine. However, this species is very unstable andtreatment with silica gel is sufficient to provoke fragmentation of theSEM protecting group accompanied by imine formation.

The SEM protected dilactams (Q) were prepared by Suzuki and Stillecoupling reactions on the enol triflate intermediate (P). The Suzukireaction is particularly useful as it can be used to install both aryland vinyl substituents at the C2 position of the PBD. In excess of 70boronic acids are commercially available allowing great diversity to beintroduced into the PBD system. Heck reactions can also be performedsmoothly on the enol triflate intermediate.

The enol triflate (P) was prepared from the ketone precursor (O) usingtriflic anhydride in DCM in the presence of pyridine. The ketone (O) wasprepared from the secondary alcohol precursor (N) by Swern oxidation.Other oxidation methods involving TPAP or the Dess Martin reagentprovide the ketone in equally good yields. The secondary alcohol wasobtained by selective removal of a TBDMS group of compound M in thepresence of the SEM N10 protecting group. The SEM group was installed byquenching the N10 dilactam anion (from L) with SEM-C1; this is a generalmethod and can be used to install related protecting groups such as MOM.In order to prevent the C2 hydroxy of compound K interfering with theN10 protection step if was protected as a TBDMS ether. The 2-hydroxydilactam (K) was formed by hydrogenating the A-ring nitro group ofcompound J and coupling to the C-ring methyl ester. The A-ring nitroC-ring ester compound (J) was prepared by coupling commerciallyavailable acid (G) to methyl 4-hydroxyprolinate.

The alternative synthesis routes are equally applicable to the synthesisof dimers.

Preferred Synthesis Strategies for Compounds of formula II

The synthesis route of scheme 1 is generally applicable to compounds offormula II.

C2-unsaturated PBDs of formula II may be synthesised from theirN10-carbamate protected precursors. Typically, palladium catalysedremoval of an allyl carbamate may be used to generate the N10-C11 iminewithout affecting the key C2-unsaturation. Alternatively, cadmium-leadcouple may be employed to cleave an N10-2,2,2-trichloroethyl carbamatefrom the protected PBD.

The reduction of the nitro-compound E as shown in scheme 1 with tin (II)chloride maintains the C2-unsaturation, although isolating the aniline Cfrom the tin salts can be problematic.

The compound of formula F may be used in its TBDMS protected form, andtherefore a deprotection step has to be included to produce theamino-alcohol compound E.

The TBDMS ether of type E, which is the product of the coupling of theTBDMS protected compound with the appropriate o-nitrobenzoyl chloride,can be treated with AcOH:THF:H₂O (3:1:1). TBAF was found to beunsuitable for this transformation due to the rapid degradation ofreaction products.

C-ring providing compounds F(II) can be obtained as shown in scheme 8.

Commercially available trans-4-hydroxy-L-proline F8 can be N-allocprotected to give the allyl carbamate F7 which can then be esterifiedusing standard conditions. Hydride reduction of the ester F6 furnishesthe diol F5. Selective TBDMS protection of the diol gives a silyl etherF4, which can then be oxidised, using either Swern or TPAP oxidation, toprovide the ketone F3.

The C2-olefinic functionality present in F2 may be introduced byperforming the Wittig reaction on ketone F3. Palladium-mediated cleavageof the N-alloc protecting group (Dangles O.; Guibé, F.; Balavoine, G.;Lavielle, S.; Marquet, A.; J. Org. Chem. 1987, 52, 4984) yields compoundF(II).Alternative Route to Compound C

An alternative route to compound C has been developed (Scheme 9). Theamide of formula C1 may be synthesised by forming the acid chloride ofan N-Troc protected anthranilic acid of type C2. Interestingly, N-Trocanthranilic acids do not generate isatoic anhydrides, thus enablingamide formation reactions with amines of type F(II). SimultaneousTBAF-mediated cleavage of the 2,2,2-trichloroethyl carbamate and TBDMSgroups from C1 may provide the key amino-alcohol C.Alternative Route to Compounds of Formula II

A more linear synthetic route to compound B of scheme 1 has beendeveloped which enables larger scale production of the C2-unsaturatedPBDs, and is shown in scheme 10. TBAF-mediated cleavage of the TBDMSgroup may be used to produce B(II) from B1(II). The key C2-unsaturationpresent in B1(II) may be introduced by performing the Wittig olefinationreaction on a ketone of type B2. Swern oxidation of the secondaryalcohol B3 may be used to furnish the ketone B2. The carbamate protectedaniline B3 may be prepared from the nitro compound B5 in two steps.Firstly, the nitro group may be reduced to the aniline by employing theRaney nickel/hydrazine method because a compound of type B5 lacksC2-unsaturation. This method is more advantageous than the tin (II)chloride procedure since the product is easier to isolate. The anilineB4 may then be N-carbamate protected in high yield without significantcarbonate formation at the C2 oxygen.

An amide of type B5 may be synthesised by coupling an acid chloride oftype G to the key amine KEC5 (see scheme 6). Overall, this route hasseveral advantages over the convergent route which allow larger scaleproduction of the C2-unsaturated PBDs. Firstly, catalytic hydrogenationof KEC4 allows large scale preparation of key intermediate KEC5.Secondly, the nitro reduction step may be carried out on an intermediatedevoid of C2-unsaturation. Finally, the Wittig olefination may beperformed in the latter stages of the synthetic route where large scalelimitations are tolerated.

In dimer synthesis, the routes set out above may be used in preferenceto those set out in the overall synthetic strategies. In particular, thenitrogen-protecting group may advantageously be a carbamate, asprotecting groups of this type may be removed in the final step by avariety of methods which, in general, do not affect the keyC2-unsaturation.

General Experimental Methods

Melting points (mp) were determined on a Gallenkamp P1384 digitalmelting point apparatus and are uncorrected. Infrared (IR) spectra wererecorded using a Perkin-Elmer 297 spectrophotometer. ¹H- and ¹³C-NMRspectra were recorded on a Jeol GSX 270 MHZ FT-NMR spectrometeroperating at 20° C.+/−1° C. Chemical shifts are reported in parts permillion (δ) downfield from tetramethylsilane (TMS). Spin multiplicitiesare described as: s (singlet), bs (broad singlet), d (doublet), dd(doublet of doublets), t (triplet), q (quartet), p (pentuplet) or m(multiplet). Mass spectra (MS) were recorded using a Jeol JMS-DX 303 GCMass Spectrometer (EI mode: 70 eV, source 117-147° C.). Accuratemolecular masses (HRMS) were determined by peak matching usingperfluorokerosene (PFK) as an internal mass marker, and FAB mass spectrawere obtained from a glycerol/thioglycerol/trifluoroacetic acid(1:1:0.1) matrix with a source temperature of 180° C. Optical rotationsat the Na-D line were obtained at ambient temperature using aPerkin-Elmer 141 Polarimeter. Analytical results were generally within+/−0.2% of the theoretical values. Flash chromatography was performedusing Aldrich flash chromatography “Silica Gel-60” (E. Merck, 230-400mesh). Thin-layer chromatography (TLC) was performed using GF₂₅₄ silicagel (with fluorescent indicator) on glass plates. All solvents andreagents, unless otherwise stated, were supplied by the Aldrich ChemicalCompany Ltd. and were used as supplied without further purification.Anhydrous solvents were prepared by distillation under a dry nitrogenatmosphere in the presence of an appropriate drying agent, and werestored over 4A molecular sieves or sodium wire. Petroleum ether refersto the fraction boiling at 40-60° C.

EXAMPLES Example 1(a)

Synthesis of the 2-Cyanomethyl PBD (10, SB-A67) (See FIG. 1)

Synthesis of the Nitro Alcohol (3)

A solution of the acid 1 (3.03 g, 10 mmol, 1 equiv) in freshly distilledCH₂Cl₂ (50 mL) was treated with oxalyl chloride (1.05 mL, 12 mmol, 1.2equiv) under a nitrogen atmosphere and stirred. DMF (0.1 mL) was addedand the solution effervesced. The reaction was allowed to stir overnightat RT. The following day the acid chloride solution was added dropwiseover 2 hours to a stirred mixture of the amine 2 (2.31 g, 10 mmol, 1equiv) and TEA (3.48 mL, 25 mmol, 2.5 equiv) in freshly distilled CH₂Cl₂(30 mL) while the temperature was kept under 0° C., under a nitrogenatmosphere. The reaction mixture was then allowed to warm to RT andstirred overnight. The solution was washed with NaHCO₃ (100 mL),saturated NH₄Cl (100 mL), H₂O (100 mL), brine (100 mL), dried (MgSO₄),filtered and evaporated in vacuo to give a brown oil which was purifiedby flash chromatography (SiO₂, EtOAc) and provided the coupled compound3 (3.24 g, 6.28 mmol, 62.8%) as a yellow glass: ¹H NMR (CDCl₃, 270 MHz)rotamers: δ−0.10 (s, 6H, Si(CH₃)₂), 0.80 (s, 9H, SiC(CH₃)₃, 2.04-2.55(m, 3H, 1-H, OH), 3.05-4.60 (m, 9H, 11-H, 11a-H, OMe, 3-H, 2-H), 5.20(br s, 2H, OBn), 6.78 and 6.85 (2×s, 1H, 6-H), 7.27-7.47 (m, 5H, Ph),7.73 and 7.76 (2×s, 1H, 9-H); ¹³C NMR (CDCl₃, 270 MHz): δ−5.5, −5.4,18.2, 25.7, 25.8, 36.3, 56.6, 57.2, 62.6, 70.2, 71.3, 109.0, 109.4,127.6-128.8, 135.2, 137.3, 147.9, 154.7, 166.6; IR (neat): 3401, 3065,3033, 2951, 2856, 2739, 2628, 1956, 1743, 1620, 1578, 1522, 1462, 1434,1378, 1336, 1277, 1221, 1075, 1051, 1002, 914, 836, 779, 752, 697, 669,650, 615; EIMS m/z (relative intensity) 516 (M^(+.) , 0.6), 460 (29.8),459 (92.6), 368 (7.9), 286 (49.6), 91 (100.0), 73 (9.5); FAB m/z(relative intensity) 517 (M^(+.) +1, 15.1), 385 (9.2), 286 (19.3), 92(9.3), 91 (100.0), 75 (14.0), 73 (42.2).

Reduction to the Amino Alcohol (4)

A solution of hydrazine (3.11 mL, 100 mmol, 5 equiv) in MeOH (50 mL) wasadded dropwise to a refluxing solution of the nitro compound 3 (10.32 g,20 mmol, 1 equiv), antibumping granules and Raney Ni (3.5 g) in MeOH(150 mL). After 1 hour at reflux TLC (SiO₂, 5% MeOH—CHCl₃) revealedtotal consumption of starting material. The mixture was then treatedwith sufficient Raney Ni to decompose any unreacted hydrazine. Aftercooling to RT the mixture was filtered through Celite and the filtrateevaporated in vacuo. The resulting residue was dissolved in CH₂Cl₂ (300mL), dried (MgSO₄), filtered and evaporated in vacuo to provide 4 (6.80g, 14 mmol, 70%) as a pink oil which was carried through to the nextstage without purification: ¹H NMR (CDCl₃, 270 MHz) rotamers: δ−0.001(s, 6H, Si(CH₃)₂), 0.88 (br s, 9H, SiC(CH₃)₃), 1.96-2.23 (m, 2H, 1-H),3.44-4.48 (m, 12H, 11-H, 3-H, OMe, NH₂, OH, 2-H, 11a-H), 5.09 (br s, 2H,OBn), 6.25 and 6.27 (2×s, 1H, 6-H), 6.68 and 6.73 (2×s, 1H, 9-H),7.26-7.42 (m, 5H, Ph); ¹³C NMR (CDCl₃, 270 MHz): δ−5.4, 18.2, 25.9,35.7, 56.9, 57.2, 70.4, 70.7, 103.2, 112.9, 113.4, 127.2, 127.4, 127.9,128.6, 128.6, 136.7, 141.6; IR (neat): 3356.80, 2930.13, 2857.36,2247.82, 1622.19, 1514.60, 1463.60, 1408.95, 1261.43, 1176.55, 1118.48,1003.88, 911.00, 836.61, 778.15, 733.59, 697.72, 646.32.

Synthesis of the Alloc Pro-N10-Protected C2-Alcohol (5)

A solution of allyl chloroformate (1.54 mL, 14.48 mmol, 1.05 equiv) infreshly distilled CH₂Cl₂ (30 mL) was added dropwise to a stirred mixtureof the amine 4 (6.70 g, 13.79 mmol, 1 equiv), pyridine (2.45 mL, 30.34mmol, 2.2 equiv) in freshly distilled CH₂Cl₂ (200 mL), at 0° C. under anitrogen atmosphere. The mixture was allowed to warm at RT and stirredovernight. The following day TLC (SiO₂, 5% MeOH—CHCl₃) revealed reactioncompletion. The mixture was washed with saturated CuSO₄ (100 mL), H₂O(100 mL), brine (100 mL), dried (MgSO₄), filtered and evaporated invacuo to give a dark yellow oil. Flash chromatography (SiO₂, 30%EtOAc-petroleum ether) afforded the pure Alloc-compound 5 (6.70 g, 11.75mmol, 85.2%) as a yellow oil: ¹H NMR (CDCl₃, 270 MHz) rotamers: δ0.03and 0.04 (2×s, 6H, Si(CH₃)₂), 0.89 (br s, 9H, SiC(CH₃)₃), 1.99-2.40 (m,2H, 1-H), 3.56 (br s, 4H, 11-H, 3-H), 3.79 (s, 3H, OMe), 4.05-4.20 (m,1H, 11a-H), 4.38 (s, 1H, 2-H), 4.58-4.62 (m, 3H, OH, Alloc), 5.16-5.37(m, 4H, OBn, Alloc), 5.86-6.00 (m, 1H, Alloc), 6.80 (s, 1H, 6-H),7.30-7.48 (m, 5H, Ph), 7.80 (s, 1H, 9-H), 8.86 (br s, 1H, NH); ¹³C NNR(CDCl₃, 270 MHz): δ−5.5, −5.4, 18.1, 25.8, 35.6, 56.4, 57.2, 60.4, 65.8,70.5, 70.7, 106.4, 111.7, 116.4, 118.0, 127.7-128.6, 132.5, 136.3,144.3, 150.2, 153.8, 169.4; IR (neat): 3336, 3067, 2953, 2931, 2858,1732, 1600, 1525, 1464, 1408, 1327, 1225, 1175, 1121, 1048, 1028, 1002,937, 837, 812, 778, 744, 698, 671, 636, 608, 562; EIMS m/z (relativeintensity) 570 (M^(+.) , 35.0), 513 (27.2), 340 (19.3), 149 (24.3), 91(24.1), 77 (16.4), 58 (33.0), 57 (100.0), 44 (27.2), 39 (39.8); [α]²³_(D)=−55.94° (c=1.010, CHCl₃).

Oxidation to the C2-Ketone (6)

A solution of DMSO (2.50 mL, 35.25 mmol, 3 equiv) in freshly distilledCH₂Cl₂ (200 mL) was added dropwise over 1.5 hours to a stirred solutionof oxalyl chloride (8.81 mL of a 2M solution in CH₂Cl₂, 17.62 mmol, 1.5equiv) at −55/−60° C. (liquid nitrogen/CHCl₃) under a nitrogenatmosphere. After 30 minutes stirring at −55° C., a solution of thesecondary alcohol 5 (6.70 g, 11.75 mmol, 1 equiv) in CH₂Cl₂ (150 mL) wasadded dropwise to the reaction mixture over 1.5 h. Following stirring at−55/−60° C. for 45 minutes the reaction was treated dropwise with asolution of TEA (11.14 mL, 79.90 mmol, 6.8 equiv) in CH₂Cl₂ (50 mL) overa period of 40 minutes. The mixture was stirred for a further 45 minutesat −30° C. and was then allowed to warm to RT. The reaction was thentreated with brine (150 mL), cooled to 0° C. and acidified to pH=2 withconcentrated HCl. The organic phase was washed with H₂O (150 mL), brine(150 mL), dried (MgSO₄), filtered and evaporated in vacuo to give theketone 6 as a dark orange oil (6.18 g, 10.88 mmol, 93%), sufficientlypure by TLC (SiO₂, 40% EtOAc-petroleum ether) to be carried through tothe next stage without further purification: ¹H NMR (CDCl₃, 270 MHz)rotamers: δ0.04 and 0.05 (2×s, 6H, Si(CH₃)₂), 0.87 (s, 9H, SiC(CH₃)₃),2.47-2.78 (m, 2H, 1-H), 3.66-4.10 (m, 8H, 3-H, OMe, 11-H, 11a-H),4.62-4.65 (m, 2H, Alloc), 4.80-5.40 (m, 4H, OBn, Alloc), 5.88-6.03 (m,1H, Alloc), 6.76 (s, 1H, 6-H), 7.27-7.49 (m, 5H, Ph), 7.90 (s, 1H, 9-H),8.62 (br s, 1H, NH); ¹³C NMR (CDCl₃, 270 MHz): δ−5.8, −5.7, 18.0, 25.6,25.7, 56.5, 65.8, 68.0, 70.7, 106.4, 111.0, 118.2, 127.7-128.6, 132.4,136.1, 150.6, 153.4, 208.9; IR (neat): 3510, 3332, 2957, 2870, 2740,1959, 1771, 1738, 1633, 1537, 1428, 1274, 1233, 1120, 1029, 844, 785,700; EIMS m/z (relative intensity) 568 (M^(+.) , 90.6), 512 (28.7), 511(79.8), 453 (12.1), 340 (38.6), 298 (12.7), 282 (16.9), 172 (23.9), 91(100.0), 41 (15.1); [α]²³ _(D)=−1.98° (c=1.010, CHCl₃).

Insertion of the C₂-Cyanomethyl Group (7)

Sodium hydride (0.70 g of a 60% dispersion inminuteseral oil, 17.60mmol, 2.5 equiv) was stirred in petroleum ether for 10 minutes. Thesuspension was allowed to settle and the solvent transferred undernitrogen from the flask via a double-tipped needle. The remainingresidue was suspended in freshly distilled anhydrous THF (50 mL), cooledto 0° C. and treated dropwise with a solution of the diethylcyanomethylphosphonate (11.14 mL, 79.90 mmol, 6.8 equiv) in THF (60 mL)under a nitrogen atmosphere. The mixture was allowed to warm to RT andstir for 1.5 h. After cooling to 0° C. the reaction mixture was treateddropwise with a solution of the ketone 6 (11.14 mL, 79.90 mmol, 6.8equiv) in THF (40 mL). After stirring overnight TLC (SiO₂, 30%EtOAc-petroleum ether) revealed almost complete consumption of startingmaterial. THF was evaporated in vacuo and the resulting residue treatedwith a saturated solution of NaHCO₃ (100 mL) and EtOAc (100 mL). Theaqueous layer was washed with EtOAc (100 mL) and the combined organiclayers were then washed with H₂O (100 mL), brine (100 mL), dried(MgSO₄), filtered and evaporated in vacuo to give a brown glass whichwas subjected to flash chromatography (SiO₂, 30% EtOAc-petroleum ether)to provide the pure cyano compound 7 (2.6 g, 4.40 mmol, 63%) as a yellowglass: ¹H NMR (CDCl₃, 270 MHz): δ0.03-0.09 (m, 6H, Si(CH₃)₂), 0.88 (m,9H, SiC(CH₃)₃), 2.68-2.91 (m, 2H, 1-H), 3.12-3.13 (m, 2H, 12-H),3.72-3.76 (m, 2H, 11-H), 3.82 (s, 3H, OMe), 4.62-4.65 (m, 2H, Alloc),4.75 (m, 1H, 11a-H), 5.19 (s, 2H, OBn), 5.22-5.39 (m, 2H, Alloc),5.88-6.02 (m, 1H, Alloc), 6.59 (s, 1H, 3-H), 6.68 (s, 1H, 6-H),7.32-7.50 (m, 5H, Ph), 7.95 (s, 1H, 9-H), 8.72 (s, 1H, NH); ¹³C NMR(CDCl₃, 270 MHz): δ−5.4, 17.5, 18.1, 25.6-25.7, 34.0, 56.6, 59.8, 62.3,65.8, 70.7, 106.1, 111.8, 114.0, 116.2, 118.1, 127.7-129.3, 132.4,132.8, 136.1, 144.2, 150.9, 153.4, 166.1; IR (neat): 3337, 3067, 3034,2954, 2930, 2857, 2253, 1732, 1622, 1599, 1524, 1495, 1464, 1408, 1362,1336, 1259, 1205, 1166, 1116, 1051, 1026, 992, 914, 839, 778, 735, 698,647; EIMS m/z (relative intensity) 591 (M^(+.) , 20.1), 534 (15.0), 340(67.5), 282 (20.9), 252 (25.6), 195 (32.4), 91 (100.0); HRMS m/z Calcdfor 591.2765 (C₃₂H₄₁N₃O₆Si). Found 591.2758; [α]²³ _(D)=−83.25°(c=1.015, CHCl₃).

Deprotected Alcohol (8)

Glacial ACOH (15 mL) was added to a stirred solution of the silyl ether7 (2.10 g, 3.55 mmol) in THF (10 mL) and H₂O (15 mL). The reactionmixture was allowed to stir at RT and monitored every hour by TLC (SiO₂,30% EtOAc-petroleum ether). Over the course of 3 hours AcOH (10 mL) wasadded in two further portions. The mixture was stirred for a total of 4hours at which time the reaction had gone to completion. The mixture wasthen cooled to 0° C. and treated dropwise with a 10% solution of NaHCO₃in H₂O (50 mL). The aqueous solution was extracted with EtOAc (3×20 mL)and the combined organic layers were washed with H₂O (20 mL), brine (20mL), dried (MgSO₄), filtered and evaporated in vacuo to give a yellowoil. Flash chromatography (SiO₂, 5% MeOH—CHCl₃) afforded the freealcohol 8 (1.40 g, 2.93 mmol, 83%) as a yellow glass: ¹H NMR (CDCl₃, 270MHz): δ2.41-3.02 (m, 2H, 1-H), 3.13 (s, 2H, 12-H), 3.70-4.10 (m, 6H,11-H, OMe, OH), 4.61-4.64 (m, 2H, Alloc), 4.76 (m, 1H, 11a-H), 5.16 (s,2H, OBn), 5.23-5.28 (m, 2H, Alloc), 5.87-6.02 (m, 1H, Alloc), 6.53 (s,1H, 3-H), 6.78 (s, 1H, 6-H), 7.27-7.48 (m, 5H, Ph), 7.75 (s, 1H, 9-H),8.45 (s, 1H, NH); ¹³C NMR (CDCl₃, 270 MHz): δ17.4, 34.8, 56.8, 61.5,65.1, 65.9, 70.8, 106.9, 111.8, 114.4, 116.1, 118.2, 127.7-129.1, 132.1,132.4, 136.0, 144.8, 151.1, 153.7, 167.3; IR (neat): 3340, 3067, 2934,2856, 2252, 1732, 1601, 1523, 1455, 1407, 1374, 1336, 1226, 1167, 1111,1048, 1028, 996, 938, 869, 838, 768, 745, 698, 668, 636, 608; EIMS m/z(relative intensity) 477 (M^(+.) , 14.6), 340 (46.9), 282 (13.0), 91(100.0); HRMS m/z Calcd for 477.1900 (C₂₆H₂₇N₃O₆. Found 477.1962; [α]²³_(D)=−67.42° (c=1.068, CHCl₃).

N10-Protected Cyclized PBD (9)

A solution of DMSO (0.75 mL, 10.55 mmol, 3.6 equiv) in freshly distilledCH₂Cl₂ (40 mL) was added dropwise at a rapid rate to a stirred solutionof oxalyl chloride (2.64 mL of a 2M solution in CH₂Cl₂, 5.27 mmol, 1.8equiv) at −40/−50° C. (liquid nitrogen/chlorobenzene) under a nitrogenatmosphere. After 5 minutes stirring at −45° C., a solution of theprimary alcohol 8 (1.40 g, 2.93 mmol, 1 equiv) in CH₂Cl₂ (30 mL) wasadded dropwise to the reaction mixture over 45 minutes. Followingstirring at −45° C. for 45 minutes the reaction was treated dropwisewith a solution of TEA (1.72 mL, 12.31 mmol, 4.2 equiv) in CH₂Cl₂ (20mL) over a period of 30 minutes. The mixture was stirred for a further40 minutes at −45° C. and was then allowed to warm to RT and dilutedwith 20 mL CH₂Cl₂. The reaction was then cooled to 0° C. and washed with1N HCl (200 mL), H₂O (100 mL), brine (100 mL), dried (MgSO₄), filteredand evaporated in vacuo to give a yellow foam which was subjected toflash chromatography (SiO₂, 5% MeOH—CHCl₃) to provide the pure ringclosed compound 9 (0.95 g, 2.00 mmol, 68%) as a slightly yellow glass:¹H NMR (CDCl₃, 270 MHz): δ2.69-3.14 (m, 2H, 1-H), 3.24 (s, 2H, 12-H),3.84-3.98 (m, 6H, 11-H, OMe, OH), 4.46 (m, 2H, Alloc), 5.07-5.18 (m, 4H,OBn, Alloc), 5.60-5.80 (m, 2H, Alloc, 11a-H), 6.74 (s, 1H, 3-H), 7.04(s, 1H, 6-H), 7.24-7.43 (m, 6H, Ph, 9-H); ¹³C NMR (CDCl₃, 270 MHz):δ17.5, 36.5, 56.2, 59.6, 66.9, 71.1, 85.7, 111.0, 113.2, 114.7, 116.1,118.3, 124.6, 127.3-128.7, 131.7, 136.0, 149.2, 150.6, 163.6; IR (neat):3396, 3089, 2938, 2615, 2251, 1707, 1602, 1513, 1432, 1308, 1219, 1113,1045, 918, 869, 790, 735, 698, 648; EIMS m/z (relative intensity) 475(M^(+.), 34.2), 340 (25.4), 339 (35.0), 279 (10.3), 134 (10.6), 91(100.0); HRMS m/z Calcd for 475.1743 (C₂₆H₂₅N₃O₆). Found 475.1883; [α]²³_(D)=+101.46° (c=1.030, CHCl₃).

C₂-Cyanomethyl PBD (10, SB-A67)

Triphenylphosphine (25 mg, 0.095 mmol, 0.05 equiv), pyrrolidine (167 μl,2.0 mmol, 1.05 equiv) and Pd(PPh₃)₄ (56 mg, 0.048 mmol, 0.025 equiv)were added sequentially to a stirred solution of the Alloc-compound 9(900 mg, 1.90 mmol, 1 equiv) in freshly distilled dry CH₂Cl₂ (100 mL).The reaction mixture was allowed to stir at RT under a nitrogenatmosphere for 2 hours by which time TLC (SiO₂, 1% MeOH—CHCl₃) revealedreaction completion. The mixture was evaporated in vacuo and the residueapplied to a gravity chromatography column (SiO₂, 1% MeOH—CHCl₃) toisolate the PBD SB-A67 (720 mg, 1.93 mmol, 100%) as a yellow glass: ¹HNMR (CDCl₃, 270 MHz): 3.05-3.40 (m, 4H, 1-H, 12-H), 3.95 (s, 3H, OMe),4.38 (m, 1H, 11a-H), 5.21 (s, 2H, OBn), 6.84 (s, 1H, 6-H), 7.06 (s, 1H,3-H), 7.27-7.70 (m, 6H, Ph, 9-H), 7.80 (d, 1H, 11a-H, J=3 Hz); ¹³C NMR(CDCl₃, 270 MHz): δ17.4, 36.8, 53.9, 56.3, 70.9, 111.7, 111.9, 112.8,116.0, 118.7, 120.7, 127.1-128.7, 132.0, 136.0, 140.2, 148.3, 151.2,161.8; IR (neat): 3353, 2931, 2251, 2222, 1604, 1508, 1437, 1247, 1120,1000, 913, 874, 724, 697, 542; EIMS m/z (relative intensity) 373(M^(+.), 9.8), 371 (24.4), 280 (12.5), 91 (100.0); HRMS m/z Calcd for373.1426 (C₂₂H₁₉N₃O₃). Found 373.1364; [α]²³ _(D)=254.5° (c=1.045,CHCl₃).

Example 1(b)

Synthesis of the 2-Methoxycarbonylmethyl PBD (24, SJG-245) (See FIG. 2)

(2S,4R)-N-(Allyloxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic Acid (12)

A solution of allyl chloroformate (29.2 mL, 33.2 g, 275 mmol) in THF (30mL) was added dropwise to a suspension of trans-4-hydroxy-L-proline (11)(30 g, 229 mmol) in a mixture of THF (150 mL) and H₂O (150 mL) at 0° C.(ice/acetone), whilst maintaining the pH at 9 with 4 N NaOH. Afterstirring at 0° C. for 1 hour at pH 9, the aqueous layer was saturatedwith NaCl, and the mixture diluted with EtOAc (100 mL). The aqueouslayer was separated, washed with EtOAc (100 mL) and the pH adjusted to 2with conc. HCl. The resulting milky emulsion was extracted with EtOAc(2×100 mL), washed with brine (200 mL), dried (MgSO₄), filtered andevaporated in vacuo to give the allyl carbamate 12 as a clear viscousoil (42.6 g, 87%): [α]²⁰ _(D)=−62.1° (c=0.69, MeOH); ¹H NMR (270 MHz,CDCl₃+DMSO-d₆) (Rotamers) δ5.98-5.81 (m, 1H, NCO₂CH₂CH═CH₂), 5.40-5.14(m, 2H, NCO₂CH₂CH═CH₂), 4.64-4.42 (m, 4H, NCO₂CH₂CH═CH₂, NCH₂CHOHCH₂ andCHCO₂H), 3.82-3.51 (m, 2H, NCH₂CHOHCH₂), 2.34-2.08 (m, 2H, NCH₂CHOHCH₂);¹³C NMR (67.8 MHz, CDCl₃+DMSO) (Rotamers) δ175.0 and 174.5 (CO₂H), 155.1and 154.6 (NC═O), 132.9 and 132.8 (NCO₂CH₂CH═CH₂), 117.6 and 116.7(NCO₂CH₂CH═CH₂), 69.5 and 68.8 (NCH₂CHOH), 65.9 and 65.8(NCO₂CH₂CH═CH₂), 58.0 and 57.7 (CHCO₂H), 55.0 and 54.5 (NCH₂CHOH), 39.3and 38.3 (NCH₂CHOHCH₂); MS (EI), m/z (relative intensity) 215 (M^(+.),10) 197 (12), 170 (M—CO₂H, 100), 152 (24), 130 (M—CO₂C₃H₅, 97), 126(34), 112 (50), 108 (58), 86 (11), 68 (86), 56 (19); IR (Neat) 3500-2100(br, OH), 2950, 1745 and 1687 (br, C═O), 1435, 1415, 1346, 1262, 1207,1174, 1133, 1082, 993, 771 cm⁻¹; exact mass calcd for C₉H₁₃NO₅ m/e215.0794, obsd m/e 215.0791.

Methyl (2S,4R)-N-(Allyloxycarbonyl)-4-hydroxypyrrolidine-2-carboxylate(13)

A catalytic amount of concentrated H₂SO₄ (4.5 mL) was added to asolution of Alloc-hydroxyproline (12) (43 g, 200 mmol) in MeOH (300 mL)at 10° C. (ice) and the reaction mixture was then heated at reflux for 2h. After cooling to room temperature the reaction mixture was treatedwith TEA (43 mL) and the MeOH evaporated in vacuo. The residue wasdissolved in EtOAc (300 mL), washed with brine (200 mL), dried (MgSO₄),filtered and concentrated in vacuo to give a viscous oil. Purificationby flash chromatography (40% EtOAc/Petroleum Ether) removed the highR_(f) impurity to provide the pure ester 13 as a transparent yellow oil(19.6 g, 43%): [α]²³ _(D)=79.0° (c=0.35, CHCl₃); ¹H NMR (270 MHz, CDCl₃)(Rotamers) δ5.98-5.78 (m, 1H, NCO₂CH₂CH═CH₂), 5.35-5.16 (m, 2H,NCO₂CH₂CH═CH₂), 4.65-4.45 (m, 4H, NCO₂CH₂CH═CH₂, NCH₂CHOHCH₂ andNCHCO₂CH₃), 3.75 and 3.72 (s×2, 3H, OCH₃), 3.70-3.54 (m, 2H,NCH₂CHOHCH₂), 3.13 and 3.01 (br s×2, 1H, OH), 2.39-2.03 (m, 2H,NCH₂CHOHCH₂); ¹³C NMR (67.8 MHz, CDCl₃) (Rotamers) δ173.4 and 173.2(CO₂CH₃), 155.0 and 154.6 (NC═O), 132.6 and 132.4 (NCO₂CH₂CH═CH₂), 117.6and 117.3 (NCO₂CH₂CH═CH₂), 70.0 and 69.2 (NCH₂CHOH), 66.2(NCO₂CH,CH═CH₂), 57.9 and 57.7 (NCHCO₂CH₃), 55.2 and 54.6 (NCH₂CHOH),52.4 (OCH₃), 39.1 and 38.4 (NCH₂CHOHCH₂); MS (EI), m/z (relativeintensity) 229 (M^(+.), 7), 170 (M—CO₂Me, 100), 144 (M—CO₂C₃H₅, 12), 126(26), 108 (20), 68 (7), 56 (8); IR (Neat) 3438 (br, OH), 2954, 1750 and1694 (br, C═O), 1435, 1413, 1345, 1278, 1206, 1130, 1086, 994, 771 cm⁻¹;exact mass calcd for C₁₀H₁₅NO₅ m/e 229.0950, obsd m/e 229.0940.

(2S,4R)-N-(Allyloxycarbonyl)-4-hydroxy-2-(hydroxymethyl)pyrrolidine (14)

A solution of the ester 13 (19.5 g, 85 mmol) in THF (326 mL) was cooledto 0° C. (ice/acetone) and treated with LiBH₄ (2.78 g, 128 mmol) inportions. The reaction mixture was allowed to warm to room temperatureand stirred under a nitrogen atmosphere for 2.5 hours at which time TLC(50% EtOAc/Petroleum Ether) revealed complete consumption of ester 13.The mixture was cooled to 0° C. and water (108 mL) was carefully addedfollowed by 2 N HCl (54 mL). After evaporation of the THF in vacuo, themixture was neutralised to pH 7 with 10 N NaOH and saturated with solidNaCl. The saturated aqueous solution was then extracted with EtOAc(5×100 mL), the combined organic layers washed with brine (200 mL),dried (MgSO₄), filtered and evaporated in vacuo to furnish the pure diol14 as a clear colourless oil (16.97 g, 99%): [α]²⁴ _(D)=−57.0° (c=0.61,CHCl₃); ¹H NMR (270 MHz, CDCl₃) δ6.01-5.87 (m, 1H, NCO₂CH₂CH═CH₂),5.36-5.20 (m, 2H, NCO₂CH₂CH═CH₂), 4.84 (br s, 1H, NCHCH₂OH), 4.60 (d,2H, J=5.31 Hz, NCO₂CH₂CH═CH₂), 4.39 (br s, 1H, NCHCH₂OH), 4.18-4.08 (m,1H, 3.35, NCH₂CHOH), 3.90-3.35 (m, 4H, NCH₂CHOH, NCHCH₂OH, and OH), 3.04(br s, 1H, OH), 2.11-2.03 (m, 1H, NCH₂CHOHCH₂), 1.78-1.69 (m, 1H,NCH₂CHOHCH₂); ¹³C NMR (67.8 MHz, CDCl₃) δ157.1 (NC═O), 132.6(NCO₂CH₂CH═CH₂), 117.7 (NCO₂CH₂CH═CH₂), 69.2 (NCH₂CHOH), 66.4 and 66.2(NCO₂CH₂CH═CH₂ and NCHCH₂OH), 59.2 (NCHCH₂OH), 55.5 (NCH₂CHOH), 37.3(NCH₂CHOHCH₂); MS (EI), m/z (relative intensity) 201 (M^(+.), 2), 170(M—CH₂OH, 100), 144 (M—OC₃H₅, 6), 126 (26), 108 (20), 68 (9); IR (Neat)3394 (br, OH), 2946, 2870, 1679 (C═O), 1413, 1339, 1194, 1126, 1054,980, 772 cm⁻¹; exact mass calcd for C₉H₁₁NO₄ m/e 201.1001, obsd m/e201.1028.

(2S,4R)-N-(Allyloxycarbonyl)-2-(tert-butyldimethylsilyloxymethyl)-4-hydroxypyrrolidine(15)

A solution of the diol 14 (16.97 g, 84 mmol) in CH₂Cl₂ (235 mL) wastreated with TEA (11.7 mL, 8.5 g, 84 mmol) and stirred for 15 minutes atroom temperature. TBDMSCl (9.72 g, 64 mmol) and DBU (16.8 mmol, 2.51 mL,2.56 g) were added and the reaction mixture stirred for a further 16hours under a nitrogen atmosphere. The reaction mixture was diluted withEtOAc (500 mL), washed with saturated NH₄Cl (160 mL), brine (160 mL),dried (MgSO₄), filtered and evaporated in vacuo to give an oil which wasa mixture of the required product (major component), unreacted diol andthe presumed disilyated compound by TLC (50% EtOAc/Petroleum Ether).Flash chromatography (20-100% EtOAc/Petroleum Ether) isolated the 3components, to provide the monosilylated compound 15 as a slightlyyellow transparent oil (13.85 g, 52%): [α]²¹ _(D)=−58.6° (c=1.14,CHCl₃); ¹H NMR (270 MHz, CDCl₃) (Rotamers) δ6.01-5.86 (m, 1H,NCO₂CH₂CH═CH₂), 5.34-5.18 (m, 2H, NCO₂CH₂CH═CH₂), 4.59-4.49 (m, 3H,NCO₂CH₂CH═CH₂ and NCHCH₂OTBDMS), 4.06-3.50 (m, 5H, NCH₂CHOH, NCH₂CHOHand NCHCH₂OTBDMS), 2.20-2.01 (m, 2H, NCH₂CHOHCH₂), 0.87 (s, 9H,SiC(CH₃)₃), 0.0 (s, 6H, Si(CH₃)₂); ¹³C NMR (67.8 MHz, CDCl₃) (Rotamers)δ155.0 (NC═O), 133.1 (NCO₂CH₂CH═CH₂), 117.6 and 117.1 (NCO₂CH₂CH═C—H₂),70.3 and 69.7 (NCH₂CHOH), 65.9 and 65. 6 (NCO₂CH₂CH═CH₂), 63.9 and 62.8(NCHCH₂OTBDMS), 57.8 and 57.4 (NCHCH₂OTBDMS), δ55.7 and 55.2 (NCH₂CHOH),37.3 and 36.6 (NCH₂CHOHCH₂), 25.9 (SiC(CH₃)₃), 18.2 (SiC(CH₃)₃), −5.5(Si(CH₃)₂); MS (EI), m/z (relative intensity) 316 (M^(+.) +1, 29), 315(M^(+.), 4), 300 (M—CH₃, 26), 284 (4), 261 (8), 260 (50), 259 (100), 258(M—OC₃H, or M—^(t)Bu, 100), 218 (13), 215 (10), 214 (52), 200 (12), 170(M—CH₂OTBDMS, 100), 156 (40), 126 (58), 115 (33), 108 (41), 75 (35); IR(Neat) 3422 (br, OH), 2954, 2858, 1682 (C═O), 1467, 1434, 1412 (SiCH₃),1358, 1330, 1255 (SiCH₃), 1196, 1180, 1120, 1054, 995, 919, 837, 776,669 cm⁻¹; exact mass calcd for C₁₅H₂₉NO₄Si m/e 315.1866, obsd m/e315.1946.

(2S)-N-(Allyloxycarbonyl)-2-(tert-butyldimethylsilyloxymethyl)-4-oxopyrrolidine(16)

Method A: A solution of DMSO (12.9 mL, 14.3 g, 183 mmol) in CH₂Cl₂ (90mL) was added dropwise to a solution of oxalyl chloride (45.1 mL of a2.0 M solution in CH₂Cl, 90.2 mmol) at −60° C. (dry ice/acetone) under anitrogen atmosphere. After stirring at −70° C. for 30 minutes, asolution of the alcohol 15 (25.8 g, 81.9 mmol) dissolved in CH₂Cl₂ (215mL) was added dropwise at −60° C. After 1.5 hours at −70° C., themixture was treated dropwise with TEA (57.2 mL, 41.5 g, 410 mmol) andallowed to warm to 10° C. The reaction mixture was treated with brine(150 mL) and acidified to pH 3 with conc. HCl. The layers were separatedand the organic phase washed with brine (200 mL), dried (MgSO₄),filtered and concentrated in vacuo to give an orange oil. Purificationby flash chromatography (40% EtOAc/Petroleum Ether) furnished the ketone16 as a pale yellow oil (24.24 g, 95%):

Method B: A solution of the alcohol 15 (4.5 g, 14.3 mmol) in CH₂Cl₂(67.5 mL) was treated with CH₃CN (7.5 mL), 4 A powdered molecular sieves(3.54 g) and NMO (2.4 g, 20.5 mmol). After 15 minutes stirring at roomtemperature, TPAP (0.24 g, 0.7 mmol) was added to the reaction mixtureand a colour change (green→black) was observed. The reaction mixture wasallowed to stir for a further 2.5 hours at which time completeconsumption of starting material was observed by TLC (50%EtOAc/Petroleum ether 40°-60°). The black mixture was concentrated invacuo and the pure ketone 16 was obtained by flash chromatography (50%EtOAc/Petroleum Ether) as a golden oil (4.1 g, 92%): [α]²² _(D)+1.25°(c=10.0, CHCl₃); ¹H NMR (270 MHz, CDCl₃) (Rotamers) δ6.0-5.90 (m, 1H,NCO₂CH₂CH═CH₂), 5.35-5.22 (m, 2H, NCO₂CH₂CH═CH₂), 4.65-4.63 (m, 2H,NCO₂CH₂CH═CH₂), 4.48-4.40 (m, 1H, NCHCH₂OTBDMS), 4.14-3.56 (m, 4H,NCH₂C═O and NCHCH₂OTBDMS), 2.74-2.64 (m, 1H, NCH₂C═OCH₂), 2.46 (d, 1H,J=18.69 Hz, NCH₂C═OCH₂), 0.85 (s, 9H, SiC(CH₃)₃), 0.0 (s, 6H, Si(CH₃)₂);¹³C NMR (67.8 MHz, CDCl₃) (Rotamers) δ210.1 (C═O), 154.1 (NC═O), 132.7(NCO₂CH₂CH═CH₂), 118.0 and 117.7 (NCO₂CH₂CH═CH₂), 66.0 and 65.8(NCO₂CH₂CH═CH₂), 65.0 (NCHCH₂OTBDMS), 55.7 (NCHCH₂OTBDMS), 53.6(NCH₂C═O), 40.8 and 40.1 (NCH₂C═OCH₂), 25.7 (SiC(CH₃)₃), 18.1(SiC(CH₃)₃), −5.7 and −5.8 (Si(CH₃)₂); MS (CI), m/z (relative intensity)314 (M^(+.)1, 100), 256 (M—OC₃H₅ or M—^(t)Bu, 65); IR (Neat) 2930, 2858,1767 (C═O), 1709 (NC═O), 1409 (SiCH₃), 1362, 1316, 1259 (SiCH₃), 1198,1169, 1103, 1016, 938, 873, 837, 778, 683 cm⁻¹; exact mass calcd forC₁₅H₂₇NO₄Si m/e 313.1710, obsd m/e 313.1714.

(2S)-N-(Allyloxycarbonyl)-2-(tert-butyldimethylsilyloxymethyl)-4-(methoxycarbonylmethyl)-2,3-dihydropyrrole(17)

Petroleum ether 40°-60° (100 mL) was added to a sample of NaH (0.80 g ofa 60% dispersion in oil, 20.12 mmol) and stirred at room temperatureunder a nitrogen atmosphere. After 0.5 hours the mixture was allowed tosettle and the Petroleum Ether was transferred from the flask via adouble-tipped needle under nitrogen. THF (100 mL) was added to theremaining residue and the mixture was cooled to 0° C. (ice/acetone). Thecool solution was treated dropwise with a solution ofmethyldiethylphosphonoacetate (3.69 mL, 4.23 g, 20.12 mmol) in THF (100mL) under nitrogen. After 1 hour at room temperature, the mixture wascooled to 0° C. and treated dropwise with a solution of the ketone 16(3.0 g, 9.58 mmol) in THF (30 mL) under nitrogen. After 16 hours at roomtemperature, TLC (50% EtOAc/Petroleum Ether) revealed the completeconsumption of ketone and further TLC (5% EtOAc/Petroleum Ether)revealed the formation of mainly the exo-product. The reaction mixturewas cooled to 0° C. (ice/acetone) and transferred via a double-tippedneedle under nitrogen to another flask containing NaH (0.40 g of a 60%dispersion in oil, 10.1 mmol) at 0° C., freshly washed as above. Thereaction mixture was maintained at 0° C., and after 40 minutes TLCrevealed the almost complete conversion to endo-product. The THF wasevaporated in vacuo and the mixture partitioned between saturated NaHCO₃(100 mL) and EtOAc (100 mL). The layers were separated and the aqueouslayer extracted with EtOAc (2×50 mL). The combined organic layers werewashed with brine (100 mL), dried (MgSO₄), filtered and concentrated invacuo to give an orange oil. Purification by flash chromatography (5%EtOAc/Petroleum Ether) furnished the endo-ester 17 (2.22 g, 63%): [α]²¹_(D)=−97.7° (c=2.78, CHCl₃); ¹H NMR (270 MHz, CDCl₃) (Rotamers) δ6.47and 6.42 (br s×2, 1H, NCH═CCH₂CO₂CH₃), 5.98-5.86 (m, 1H, NCO₂CH₂CH═CH₂),5.31 (d, 1H, J=16.85 Hz, NCO₂CH₂CH═CH₂), 5.22 (d, 1H, J=10.62 Hz,NCO₂CH₂CH═CH₂), 4.65-4.49 (m, 2H, NCO₂CH₂CH═CH₂), 4.37-4.18 (m, 1H,NCHCH₂OTBDMS), 3.76-3.69 (m, 5H, NCHCH₂OTBDMS and CO₂H₃), 3.09 (br s,2H, NCH═CCH₂CO₂CH₃), 2.86-2.80 (m, 1H, NCH═CCH₂C₂OCH₃CH₂), 2.59 (d, 1H,J=17.40 Hz, NCH═CCH₂CO2CH₃CH₂), 0.87 (s, 9H, SiC(CH₃)₃), 0.04 and 0.03(s×2, 6H, Si(CH₃)₂); ¹³C NMR (67.8 MHz, CDCl₃) (Rotamers) δ171.2(CO₂CH₃), 151.9 (NC═O), 132.8 (NCO₂CH₂CH═CH₂), 127.1 and 126.4(NCH═CCH₂CO₂CH₃), 118.0 and 117.7 (NCO₂CH₂CH═CH₂), 114.6(NCH═CCH₂,CO₂H₃), 65.9 (NCO₂CH₂CH═CH₂), 63.4 and 62.6 (NCHCH₂OTBDMS),59.0 and 58.7 (NCHCH₂OTBDMS), 51.9 (CO2CH₃), 36.0 and 34.8(NCH═CCH₂CO₂CH₃CH₂), 34.2 and 33.9 (NCH═CCH₂COCH₃), 25.8 (SiC(CH₃)₃),18.2 (SiC(CH₃)₃), −5.4 and −5.5 (Si(CH₃)₂); MS (EI), m/z (relativeintensity) 369 (M^(+.), 58), 354 (28), 326 (31), 312 (M—OC₃H, orM—^(t)Bu, 100), 268 (80), 236 (21), 227 (86), 210 (22), 192 (22), 168(93), 152 (55), 138 (22), 120 (79), 89 (70), 73 (75); IR (NEAT) 3086,2954, 2930, 2885, 2857, 1744, 1709, 1670, 1463, 1435, 1413, 1362, 1337,1301, 1253, 1195, 1107, 1064, 1014, 983, 937, 887, 838, 778, 758, 680,662 555 cm⁻¹; exact mass calcd for C₁₈H₃₁NO₅Si m/e 369.1972, obsd m/e369.1868.

(2S)-2-(tert-butyldimethylsilyloxymethyl)-4-(methoxycarbonylmethyl)-2,3-dihydropyrrole(18)

A catalytic amount of PdCl₂(PPh₃)₂ (84 mg, 0.12 mmol) was added to astirred solution of the allyl carbamate 17 (1.10 g, 2.98 mmol) and H₂O(0.32 mL, 17.8 mmol) in CH₂Cl₂ (36 mL) After 5 minutes stirring at roomtemperature, Bu₃SnH (0.89 mL, 0.96 g, 3.30 mmol) was added rapidly inone portion. A slightly exothermic reaction with vigorous gas evolutionimmediately ensued. The mixture was stirred for 16 hours at roomtemperature under nitrogen at which time TLC (50% EtOAc/Petroleum Ether)revealed the formation of amine along with the complete consumption ofstarting material. After diluting with CH₂Cl₂ (30 mL), the organicsolution was dried (MgSO₄), filtered and evaporated in vacuo to give anorange oil which was purified by flash chromatography (50%EtOAc/Petroleum Ether) to afford the enamine 18 as a slightly orange oil(0.57 g, 67%): ¹H NMR (270 MHz, CDCl₃) δ7.53 and 7.48 (br s×2, 1H,NCH═CCH₂CO₂CH₃), 4.35-4.13 (m, 1H, NCHCH₂OTBDMS), 3.82-3.17 (m, 7H,NCH═CCH₂CO₂CH₃, NCHCH₂OTBDMS and CO₂CH₃), 2.64-2.04 (m, 2H,NCH═CCH₂CO₂CH₃CH₂), 0.90-0.88 (m, 9SiC(CH₃)₃), 0.09-0.00 (m, 6H,Si(CH₃)₂); MS (EI), m/z (relative intensity) 285 (M^(+.), 1), 270(M—CH₃, 7), 254 (6), 242 (4), 230 (6), 228 (M—^(t)Bu, 100), 212 (4), 196(3), 168 (13), 115 (3), 89 (10), 80 (4), 73 (13); MS (CI), m/z (relativeintensity) 342 (M^(+.)57, 7), 302 (M^(+.) +17, 7), 286 (M^(+.) +1, 100),228 (M—^(t)Bu, 100).

(2S)-N-(4-Benzyloxy-5-methoxy-2-nitrobenzoyl)-2-(tert-butyldimethylsilyloxymethyl)-4-(methoxycarbonylmethyl)-2,3-dihydropyrrole(19)

A catalytic amount of DMF (2 drops) was added to a stirred solution ofthe acid 1 (0.506 g, 1.67 mmol) and oxalyl chloride (0.17 mL, 0.25 g,1.98 mmol) in CH₂Cl₂ (33 mL). After 16 hours at room temperature theacid chloride solution was added dropwise to a stirred mixture of theenamine 18 (0.524 g, 1.84 mmol) and TEA (0.47 g, 0.65 mL, 4.60 mmol) inCH₂Cl₂ (12 mL) at 0° C. (ice/acetone) under a nitrogen atmosphere. Thereaction mixture was allowed to warm to room temperature and stirred fora further 2.5 h. The mixture was diluted with CH₂Cl₂ (50 mL), washedwith saturated NaHCO₃ (50 mL), saturated NH₄Cl (50 mL), H₂O (50 mL),brine (50 mL), dried (MgSO₄), filtered and evaporated in vacuo to givethe crude product as a dark orange oil. Purification by flashchromatography (25% EtOAc/Petroleum Ether) isolated the pure enamide 19as an orange oil (0.55 g, 58%): ¹H NMR (270 MHz, CDCl₃) δ7.77 (s,1H_(arom)), 7.45-7.28 (m, 5H_(arom)), 6.81 (s, 1H_(arom)), 5.80 (S, 1H,NCH═CCH₂CO₂CH₃), 5.22 (s, 2H, PhCH₂O), 4.76-4.64 (m, 1H, NCHCH₂OTBDMS),3.97 (s, 3H, OCH₃), 3.72-3.66 (m, 5H, NCHCH₂OTBDMS and CO₂CH₃), 3.02 (s,2H, NCH═CCH₂CO₂CH₃), 3.01-2.63 (m, 2H, NCH═CCH₂CO₂CH₃CH₂), 0.90 (s, 9H,SiC(CH₃)₃), 0.11 (s, 6H, Si(CH₃)₂); ¹³C NMR (67.8 MHz, CDCl₃) δ170.7(CO₂CH₃), 154.6 (NC═O), 148.3 (C_(arom)), 137.6 (C_(arom)), 135.2(C_(arom)), 128.8, 128.5 and 127.6 (BnC—H_(arom)), 126.7 (C_(arom)),126.1 (NCH═CCH₂CO₂CH₃), 118.8 (NCH═CCH₂CO₂CH₃), 109.9 (C—H_(arom)),109.0 (C—H_(arom)) 71.3 (PhCH₂O), 60.7 (NCHCH₂OTBDMS), 59.0(NCHCH₂OTBDMS), 56.7 (OCH3), 52.0 (CO₂CH₃), 35.1 (NCH═CCH₂CO₂CH₃), 33.8(NCH═CCH₂CO₂CH₃CH₂), 25.8 (SiC(CH₃)₃), 18.2 (SiC(CH₃)₃), −5.3 and −5.4(Si(CH₃)₂).

(2S)-N-(4-Benzyloxy-5-methoxy-2-nitrobenzoyl)-2-(hydroxymethyl)-4-(methoxycarbonylmethyl)-2,3-dihydropyrrole(20)

A solution of the silyl protected compound 274 (0.45 g, 0.79 mmol) inTHF (8 mL) was treated with H₂O (8 mL) and glacial acetic acid (24 mL).After 5 hours stirring at room temperature TLC (50% EtOAc/PetroleumEther) showed the complete consumption of starting material. The mixturewas carefully added dropwise to a stirred solution of NaHCO₃ (64 g) inH₂O (640 mL) and extracted with EtOAc (3×100 mL). The combined organiclayers were washed with H₂O (100 mL), brine (100 mL), dried (MgSO₄),filtered and concentrated in vacuo to give the crude product as anorange glass. Purification by flash chromatography (80% EtOAc/PetroleumEther) furnished the pure alcohol 20 as a light orange glass (0.35 g,98%): ¹H NMR (270 MHz, CDCl₃) δ7.78 (s, 1H_(arom)), 7.48-7.33 (m,5H_(arom)), 6.86 (s, 1H_(arom)), 5.82 (s, 1H, NCH═CCH₂CO₂CH₃), 5.22 (s,2H, PhCH₂O), 4.81-4.71 (m, 1H, NCHCH₂OH), 3.98-3.92 (m, 5H, NCHCH₂OH andOCH₃), 3.72 (s, 3H, CO₂CH₃), 3.10-2.22 (m, 3H, NCH═CCH₂CO₂CH₃ andNCH═CCH₂CO₂CH₃CH₂), 2.50-2.35 (m, 1H, NCH═CCH₂CO₂CH₃CH₂); ¹³C NMR (67.8MHz, CDCl₃) δ170.6 (CO₂CH₃), 154.8 (NC═O), 148.5 (C_(arom)), 137.5(C_(arom)), 135.1 (C_(arom)), 128.9, 128.6 and 127.6 (BnC—H_(arom)),126.2 (NCH═CCH₂CO₂CH₃), 119.4 (NCH═CCH₂CO₂CH₃), 109.8 (C—H_(arom)),109.0 (C—H_(arom)), 71.4 (PhCH₂O), 61.5 (NCHCH₂OH), 61.4 (NCHCH₂OH),56.8 (OCH₃), 52.1 (CO₂CH₃), 35.6 (NCH═CCH₂CO₂CH₃), 33.5(NCH═CCH₂CO₂CH₃CH₂); MS (EI), m/z (relative intensity) 456 (M^(+.), 7),286 (M—NCHC═CH₂CO₂CH₃CH₂CHCH₂OH, 25), 270 (NCHC═CH₂CO₂CH₃CH₂CHCH₂OH, 6),91 (PhCH₂, 100), 80 (6); exact mass calcd for C₂₃H₂₁N₂O₈ m/e 456.1533,obsd m/e 456.1557.

(2S)-N-(2-Amino-4-benzyloxy-5-methoxybenzoyl)-2-(hydroxymethyl)-4-(methoxycarbonylmethyl)-2,3-dihydropyrrole(21)

A solution of the nitro-alcohol 20 (0.35 g, 0.77 mmol) and SnCl₂/2H₂O(0.87 g, 3.86 mmol) in methanol (16 mL) was heated to reflux andmonitored by TLC (90% CHCl₃/MeOH). After 1 hour the MeOH was evaporatedin vacuo and the resulting residue cooled (ice), and treated carefullywith saturated NaHCO₃ (65 mL). The mixture was diluted with EtOAc (65mL), and after 16 hours stirring at room temperature the inorganicprecipitate was removed by filtration through celite. The organic layerwas separated, washed with brine (100 mL), dried (MgSO₄), filtered andevaporated in vacuo to give the crude amine 21 as a pale orange glass(0.29 g, 88%) which was carried through to the next step without furtherpurification or analysis due to the instability of the amine.

(2S)-N-[(2-Allyloxycarbonylamino)-4-benzyloxy-5-methoxybenzoyl]-2-(hydroxymethyl)-4-(methoxycarbonylmethyl)-2,3-dihydropyrrole(22)

A solution of the amino-alcohol 21 (0.29 g, 0.68 mmol) in CH₂Cl₂ (12 mL)was cooled to 0° C. (ice/acetone) and treated with pyridine (0.11 mL,0.11 g, 1.39 mmol). A solution of allyl chloroformate (79 μL, 90 mg,0.75 mmol) in CH₂Cl₂ (10 mL) was then added dropwise to the stirredmixture. The reaction mixture was allowed to warm to room temperatureand stirred for a further 2.5 h, at which point TLC (EtOAc) revealedcomplete consumption of the amine 21. The mixture was diluted withCH₂Cl₂ (30 mL) and washed with saturated CuSO₄ (20 mL), H₂O (20 mL),brine (20 mL), dried (MgSO₄), filtered and evaporated in vacuo. Thecrude residue was purified by flash chromatography (70% EtOAc/PetroleumEther) to afford the pure alloc-amino compound 22 as a colourless glass(0.14 g, 40%): ¹H NMR (270 MHz, CDCl₃) δ8.58 (br s, 1H, NH), 7.88 (br s,1H_(arom)), 7.50-7.29 (m, 5H_(arom)), 6.83 (s, 1H_(arom)), 6.42 (br s,1H, NCH═CCH₂CO₂CH₃), 6.03-5.89 (m, 1H, NCO₂CH₂CH═CH₂), 5.39-5.22 (m, 2H,NCO₂CH₂CH—CH₂), 5.18 (s, 2H, PhCH₂O), 4.77-4.73 (m, 1H, NCHCH₂OH),4.65-4.62 (m, 2H, NCO₂CH₂CH═CH₂), 4.32-3.84 (m, 5H, NCHCH₂OH and OCH₃),3.69 (s, 3H, CO₂CH₃), 3.09 (s, 2H, NCH═CCH₃CO₂CH₃), 3.05-2.95 (m, 1H,NCH═CCH₂CO₂CH₃CH₂), 2.35 (dd, 1H, J=3.76, 16.72 Hz, NCH═CCH₂CO₂CH₃CH₂);¹³C NMR (67.8 MHz, CDCl₃) δ170.6 (CO₂CH₃), 167.4 (NC═O_(amide)), 153.5(NC═O_(carbamate)), 151.1 (C_(arom)), 144.4 (C_(arom)), 136.1(C_(arom)), 132.6 (C_(arom)), 132.4 (NCO₂CH₂CH═CH₂), 128.6, 128.1 and127.7 (BnC—H_(arom)), 118.5 (NCH═CCH₂CO₂CH₃), 118.2 (NCO₂CH₂CH═CH₂),112.1 (C—H_(arom)), 106.3 (C—H_(arom)), 70.7 (PhCH₂O), 66.5 (NCHCH₂OH),65.9 (NCO₂CH₂CH═CH₂), 61.9 (NCHCH₂OH), 56.7 (OCH3), 52.1 (CO₂CH₃), 35.6(NCH═CCH₂CO₂CH₃), 33.6 (NCH═CCH₂CO₂CH₃CH₂); MS (FAB), m/z (relativeintensity) 618 (M^(+.)+Thioglycerol, 2), 511 (M^(+.) +1, 5), 510(M^(+.), 1), 340 (M—NCH═CCH₂CO₂CH₃CH₂CHCH₂OH, 20), 300 (3), 282 (14),256 (7), 192 (6), 171 (16), 149 (22), 140 (12), 112 (4), 91 (PhCH₂,100), 80 (6), 65 (1), 57 (3).

(11S,11aS)-10-Allyloxycarbonyl-8-benzyloxy-11-hydroxy-7-methoxy-2-(methoxycarbonylmethyl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(23)

A solution of the alcohol 22 (0.14 g, 0.28 mmol) in CH₂Cl₂/CH₃CN (12 mL,3:1) was treated with 4 Å powdered molecular sieves (0.15 g) and NMO (49mg, 0.42 mmol). After 15 minutes stirring at room temperature, TPAP(4.90 mg, 14 μmol) was added and stirring continued for a further 1 hour30 minutesutes at which point TLC (80% EtOAc/Petroleum Ether) showedproduct formation along with some unoxidised starting material. Themixture was then treated with a further quantity of NMO (49 mg, 0.42mmol) and TPAP (4.9 mg, 14 μmol), and allowed to stir for a further 0.5hours when TLC revealed reaction completion. The mixture was evaporatedin vacuo onto silica and subjected to flash chromatography (60%EtOAc/Petroleum Ether) to provide the protected carbinolamine 23 as acolourless glass (39 mg, 28%): ¹H NMR (270 MHz, CDCl₃) δ7.43-7.25 (m,6H_(arom)), 6.90 (br s, 1H_(arom)) 6.74 (s, 1H, NCH═CCH₂CO₂CH₃),5.79-5.64 (m, 1H, NCO₂CH₂CH═CH₂), 5.77 (d, 1H, J=10.26 Hz, NCHCHOH),5.19-5.06 (m, 4H, NCO₂CH₂CH═CH₂ and PhCH₂O), 4.64-4.45 (m, 2H,NCO₂CH₂CH═CH₂), 4.18-3.83 (m, 4H, OCH₃ and NCHCHOH), 3.71 (s, 3H,CO₂CH₃), 3.19 (s, 2H, NCH═CCH₂CO₂CH₃), 3.09 (dd, 1H, J=11.09, 16.70 Hz,NCH═CCH₂CO₂CH₂CH₂), 2.74 (d, 1H, J=17.03 Hz, NCH═CCH₂CO₂CH₃CH,); ¹³C NMR(67.8 MHz, CDCl₃) δ170.7 (CO₂CH₃), 163.3 (NC═O_(amide)) 155.9(NC═O_(carbamate)), 150.3 (C_(arom)), 149.1 (C_(arom)), 136.1(C_(arom)), 131.8 (NCO₂CH₂CH═CH₂), 128.7, 128.2 and 127.3(BnC—H_(arom)), 126.2 (NCH═CCH₂CO₂CH₃), 125.1 (C_(arom)), 118.1(NCO₂CH₂CH═CH₂), 117.7 (NCH═CCH₂CO₂CH₃), 114.7 (C—H_(arom)), 111.0(C—H_(arom)), 85.9 (NCHCHOH), 71.1 (PhCH₂O), 66.8 (NCO₂CH,CH═CH₂), 59.5(NCHCHOH), 56.2 (OCH₃), 52.1 (CO₂CH₃), 37.0 (NCH═CCH₂CO₂CH₃), 33.7(NCH═CCH₂CO₂CH₃CH₂); MS (EI), m/z (relative intensity) 508 (M^(+.), 16),449 (3), 422 (3), 404 (2), 368 (3), 340 (19), 324 (2), 282 (6), 255 (2),225 (1), 206 (2), 192 (3), 169 (4), 152 (2), 140 (10), 131 (5), 108 (5),91 (PhCH₂, 100), 80 (9), 57 (9), IR (NUJOL®) 3600-2500 (br, OH), 2924,2853, 2360, 1715, 1602, 1514, 1462, 1377, 1271, 1219, 1169, 1045, 722,699; exact mass calcd for C₂₇H₂₈N₂O, m/e 508.1846, obsd m/e 508.1791.

(11S,11aS)&(11R,11aS)-8-Benzyloxy-7,11-dimethoxy-2-(methoxycarbonylmethyl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(24, SJG-245)

A catalytic amount of tetrakis(triphenylphosphine)palladium (5.0 mg,4.33 μmol) was added to a stirred solution of the Alloc-protectedcarbinolamine 23 (88 mg, 0.17 mmol), triphenylphosphine (2.27 mg, 8.65μmol) and pyrrolidine (13 mg, 0.18 mmol) in CH₂Cl₂ (15 mL). After 2hours stirring at room temperature under a nitrogen atmosphere, TLC (80%EtOAc/Petroleum Ether) revealed the complete consumption of startingmaterial. The solvent was evaporated in vacuo and the crude residue waspurified by flash chromatography (60% EtOAc/Petroleum Ether) to affordthe novel PBD (SJG-245) as a colourless glass (54 mg, 77%) which wasrepeatedly evaporated in vacuo with CHCl₃ in order to provide theN10-C11 imine form 24: ¹H NMR (270 MHz, CDCl₃) (imine) δ7.80 (d, 1H,J=4.03 Hz, HC═N), 7.50 (s, 1H_(arom)), 7.45-7.26 (m, 5H_(arom)), 6.91(br s, 1H, NCH═CCH₂OC₂CH₃), 6.83 (s, 1H_(arom)), 5.21-5.12 (m, 2H,PhCH₂O), 3.94 (s, 3H, OCH₃), 3.73 (s, 3H, CO₂CH₃), 3.23 (s, 2H,NCH═CCH₂CO₂CH₃), 3.15 (m, 2H, NCH═CCH₂CO₂CH₃CH₂); ¹³C NMR (67.8 MHz,CDCl₃) (imine) δ170.7 (CO₂CH₃), 162.7 (HC═N), 161.4 (NC═O), 150.9(C_(arom)), 148.1 (C_(arom)), 140.1 (C_(arom)), 136.0 (C_(arom)), 128.7,128.2 and 127.3 (BnC—H_(arom)), 127.3 (NCH═CCH₂CO₂CH₃), 119.2(C_(arom)), 117.5 (NCH═CCH₂CO₂CH₃), 111.8 (C—H_(arom)), 111.5(C—H_(arom)), 70.8 (PhCH₂O), 56.2 (OCH₃), 53.8 (NCHHC═N), 52.0 (CO₂CH₃),37.4 (NCH═CCH₂CO₂CH₃), 33.6 (NCH═CCH₂CO₂CH₃CH₂)

Repeated evaporation in vacuo of 24 with CH₃OH provided the N10-C11methyl ether forms 25: ¹H NMR (270 MHz, CD₃OD) (11S,11aS isomer)δ7.44-7.25 (m, 5H_(arom)), 7.16 (s, 1H_(arom)), 6.85 (br s, 1H,NCH═CCH₂CO₂CH₃), 6.62 (s, 1H_(arom)), 5.09 (s, 2H, PhCH₂O), 4.52 (d, 1H,J=8.80 Hz, NCHCHOCH₃), 4.00-3.85 (m, 1H, NCHCHOCH₃), 3.80 (s, 3H, OCH₃),3.69 (s, 3H, CO₂CH₃), 3.41 (s, 3H, NCHCHOCH₃), 3.24 (br s, 2H,NCH═CCH₂CO₂CH₃), 3.20-3.00 (m, 1H, NCH═CCH₂COCH₃CH₂), 2.60-2.50 (m, 1H,NCH═CCH₂CO₂CH₃CH₂); ¹³C NMR (67.8 MHz, CD₃OD) (11S,11aS isomer) δ172.7(CO₂CH₃), 166.8 (C_(arom)), 153.3 (NC═O), 146.4 (C_(arom)), 139.7(C_(arom)), 138.0 (C_(arom)), 132.4 (C_(arom)), 129.6, 129.1 and 128.8(BnC—H_(arom)), 127.0 (NCH═CCH₂CO₂CH₃), 120.8 (NCH═CCH₂CO₂CH₃), 113.7(C—H_(arom)), 109.2 (C—H_(arom)), 97.1 (NCHCHOCH₃), 71.7 (PhCH₂O), 60.2(NCHCHOCH₃), 56.8 (OCH₃), 55.2 (NCHCHOCH₃), 52.5 (CO₂CH₃), 38.7(NCH═CCH₂CO₂CH₃), 34.1 (NCH═CCH₂CO₂CH₃CH₂); MS (EI), m/z (relativeintensity) 420 (M^(+.), methyl ether, 1), 418 (methyl ether −2, 2), 406(M^(+.), imine, 23), 404 (41), 375 (2), 345 (6), 333 (7), 313 (22), 299(10), 285 (6), 253 (6), 242 (4), 225 (2), 214 (2), 198 (2), 183 (4), 168(2), 155 (6), 136 (3), 105 (3), 91 (PhCH₂, 100), 80 (4), 65 (7); IR(NUJOL®) 3318 (br, OH of carbinolamine form), 2923, 2853, 1737, 1692,1658, 1627, 1601, 1552, 1511, 1501, 1464, 1461, 1452, 1378, 1244, 1072,1006, 786, 754, 698 cm⁻¹; exact mass calculated for C₂₃H₂₂N₂O₅ m/e406.1529, obsd m/e 406.1510.

Examples 1(c & d)

Synthesis of SJG-301 (31, UP2051) and SJG-303 (33, UP2052) (See FIG. 3)

Example 1(c) Example 1(d)

(2S)-N-[(2-Allyloxycarbonylamino)-4-benzyloxy-5-methoxybenzoyl]-2-(tert-butyldimethylsilyloethythyl)-4-(methoxycarbonylmethyl)-2,3-dihydropyrrole(26)

Petroleum Ether (100 mL) was added to a sample of NaH (1.41 g of a 60%dispersion in oil, 35.25 mmol) and stirred at room temperature under anitrogen atmosphere. After 0.5 hours the mixture was allowed to settleand the Petroleum Ether was transferred from the flask via adouble-tipped needle under nitrogen. THF (80 mL) was added to theremaining residue and the mixture was cooled to 0° C. (ice/acetone). Thecool solution was treated dropwise with a solution ofmethyldiethylphosphonoacetate (6.47 mL, 7.41 g, 35.25 mmol) in THF (80mL) under nitrogen. After 1.5 hours at room temperature, the mixture wascooled to 0° C. and treated dropwise with a solution of the ketone 6(8.0 g, 14.1 mmol) in THF (50 mL) under nitrogen. After 16 hours at roomtemperature, TLC (20% EtOAc/Petroleum Ether) revealed reactioncompletion. The THF was evaporated in vacuo and the mixture partitionedbetween saturated NaHCO₃ (200 mL) and EtOAc (220 mL). The layers wereseparated and the aqueous layer extracted with EtOAc (2×200 mL). Thecombined organic layers were washed with H₂O (200 mL), brine (200 mL),dried (MgSO₄), filtered and concentrated in vacuo to give a dark redoil. Purification by flash chromatography (15% EtOAc/Petroleum Ether)furnished the endo-ester 26 (7.02 g, 80%): [α]²² _(D)=−93.0° (c=1.04,CHCl₃); ¹H NMR (270 MHz, CDCl₃) δ8.78 (br s, 1H), 7.95 (s, 1H),7.50-7.29 (m, 5H), 6.82 (s, 1H), 6.46 (br s, 1H), 6.02-5.88 (m, 1H),5.35 (dd, 1H, J=2.93, 17.22 Hz), 5.24 (d, 1H, J=10.44 Hz), 5.18 (s, 2H),4.70-4.61 (m, 3H), 3.96-3.82 (m, 5H), 3.68 (s, 3H), 3.08 (s, 2H),2.91-2.82 (m, 1H), 2.71-2.65 (m, 1H), 0.88 (s, 9H), 0.06 and 0.04 (s×2,6H); ¹³C NMR (67.8 MHz, CDCl₃) δ170.7, 165.8, 153.5, 150.6, 144.0,136.2, 132.7, 132.5, 128.6, 128.2, 128.1, 127.7, 118.1, 118.0, 114.4,112.0, 106.0, 70.6, 65.7, 62.3, 59.4, 56.6, 52.0, 34.6, 33.9, 25.8,18.1, −5.4; MS (EI), m/z (relative intensity) 626 (M^(+.) +1, 3), 625(M^(+.) +1, 7), 624 (M^(+.), 14), 568 (5), 567 (11), 509 (3), 476 (3),341 (5), 340 (17), 339 (4), 299 (3), 286 (18), 285 (87), 282 (11), 256(4), 242 (3), 229 (3), 228 (14), 226 (11), 168 (10), 166 (3), 152 (6),141 (5), 140 (50), 139 (9), 108 (3), 92 (10), 91 (100), 89 (6), 80 (11),75 (11), 73 (10), 65 (5), 57 (6), 41 (12); IR (NEAT) 3332 (br, NH),3019, 2953, 2930, 2857, 1733, 1622, 1599, 1524, 1491, 1464, 1408, 1362,1335, 1258, 1205, 1171, 1113, 1051, 1027, 938, 839, 757, 697, 666 cm⁻¹;exact mass calcd for C₃₃H₄₄N₂O₈Si m/e 624.2867, obsd m/e 624.2936.

(2S)-N-[(2-Allyloxycarbonylamino)-4-benzyloxy-5-methoxybenzoyl]-2-(tert-butyldimethylsilyloxymethyl)-4-(hydroxy-2-ethyl)-2,3-dihydropyrrole(27)

A solution of the ester 26 (4.0 g, 6.41 mmol) in THF (55 mL) was cooledto 0° C. (ice/acetone) and treated with LiBH₄ (0.21 g, 9.62 mmol) inportions. The mixture was allowed to warm to room temperature andstirred under a nitrogen atmosphere for 26 hours at which point TLC (50%EtOAc/Petroleum Ether) revealed the complete consumption of startingmaterial. The mixture was cooled to 0° C. (ice/acetone) and water (14mL) was carefully added. Following evaporation of the THF in vacuo, themixture was cooled and then neutralised with 1 N HCl. The solution wasthen diluted with H₂O (100 mL) and extracted with EtOAc (3×100 mL), thecombined organic layers washed with brine (100 mL), dried (MgSO₄),filtered and evaporated in vacuo. The crude oil was purified by flashchromatography (30→40% EtOAc/Petroleum Ether) to furnish the pureendo-alcohol 27 as a transparent yellow oil (2.11 g, 55%): [α]²² _(D)−86.43° (c=1.38, CHCl₃); ¹H NMR (270 MHz, CDCl₃) δ8.76 (br s, 1H), 7.92(br s, 1H), 7.50-7.28 (m, 5H), 6.82 (s, 1H), 6.36 (br s, 1H), 6.02-5.87(m, 1H), 5.35 (d, 1H, J=17.22 Hz), 5.24 (d, 1H, J=11.72 Hz), 5.18 (s,2H), 4.64-4.61 (m, 3H), 4.10-3.99 (m, 1H), 3.80 (s, 3H), 3.79-3.66 (m,3H), 2.85-2.75 (m, 1H), 2.64-2.60 (m, 1H), 2.30 (t, 2H, J=6.23 Hz), 1.74(br s, 1H), 0.88 (s, 9H), 0.06 and 0.04 (s×2, 6H); ¹³C NMR (67.8 MHz,CDCl₃) δ165.3, 153.5, 150.5, 144.2, 136.3, 132.5, 128.6, 128.1, 127.7,126.7, 122.8, 118.0, 114.3, 112.0, 106.1, 70.7, 65.7, 62.8, 60.4, 59.1,56.6, 34.4, 31.7, 25.8, 18.2, −5.4; MS (EI), m/z (relative intensity)598 (M^(+.)2, 3), 597 (M^(+.) +1, 5), 596 (M^(+.), 13), 581 (2), 541(2), 540 (4), 539 (9), 448 (2), 341 (2), 340 (12), 282 (7), 259 (5), 258(20), 257 (100), 256 (3), 227 (3), 226 (12), 200 (5), 168 (6), 124 (3),113 (3), 112 (50), 111(4), 94 (10), 91 (25), 73 (3); IR (NEAT) 3340(br), 3066, 3033, 2930, 2857, 1732, 1598, 1520, 1456, 1409, 1328, 1205,1166, 1113, 1049, 1023, 938, 839, 778, 744, 697, 677, 637 cm⁻¹.

(2S)-N-[(2-Allyloxycarbonylamino)-4-benzyloxy-5-methoxybenzoyl]-4-(acyloxy-2-ethyl)-2-(tert-butyldimethylsilyloxymethyl)-2,3-dihydropyrrole(28)

Acetic anhydride (8.17 g, 7.55 mL, 80 mmol) and pyridine (30.2 mL) wereadded to the alcohol 27 (0.953 g, 1.60 mmol) and the solution stirredfor 16 hours under nitrogen at which point TLC revealed reactioncompletion (50% EtOAc/Petroleum Ether). The reaction mixture was cooledto 0° C. (ice/acetone) and treated dropwise with MeOH (15 mL). Afterstirring at room temperature for 1 hour the mixture was treated dropwisewith H₂O (30.2 mL) and allowed to stir for a further 16 h. Followingdilution with EtOAc (56 mL), the solution was cooled to 0° C. andtreated dropwise with 6 N HCl (56 mL). The layers were separated and theorganic phase was washed with 6N HCl (2×28 mL) and the combined aqueouslayers were then extracted with EtOAc (70 mL). The combined organicphases were then washed with H₂O (60 mL), brine (60 mL), dried (MgSO₄),filtered and evaporated in vacuo. The crude oil was a mixture of thedesired product 28 and the TBDMS cleaved compound 29 as judged by TLC.Purification by flash chromatography (20→100% EtOAc/Petroleum Ether)provided 29 (0.2 g) and desired acyl-TBDMS compound 28 (0.59 g, 58%) asa colourless oil: [α]²² _(D)=−87.04° (c=4.91, CHCl₃); ¹H NMR (270 MHz,CDCl₃) (Rotamers) δ8.77 (br s, 1H), 7.94 (br s, 1H), 7.49-7.31 (m, 5H),6.80 (s, 1H), 6.37 (br s, 1H), 6.02-5.89 (m, 1H), 5.35 (dd, 1H, J=17.22,1.65 Hz), 5.24 (d, 1H, J=10.30 Hz), 5.19 (s, 2H), 4.64-4.61 (m, 3H),4.12 (t, 2H, J=6.78 Hz), 4.03-3.95 (m, 1H), 3.83-3.75 (m, 4H), 2.85-2.75(m, 1H), 2.64-2.60 (m, 1H), 2.40-2.26 (m, 2H,), 2.03 (s, 3H), 0.88 (s,9H), 0.04, 0.01 and −0.01(s×3, 6H); ¹³C NMR (67.8 MHz, CDCl₃) δ170.9,165.5, 153.5, 150.6, 144.1, 136.3, 132.7, 132.5, 128.6, 128.1, 127.7,126.5, 122.2, 118.0, 114.3, 112.2, 106.1, 70.7, 65.7, 62.4, 60.4, 59.2,56.7, 34.6, 31.7, 27.9, 25.8, 20.9, 18.2, −5.4; MS (EI), m/z (relativeintensity) 640 (M^(+.) +2, 3), 639 (M^(+.) +1, 7), 638 (M^(+.), 15), 623(2), 583 (3), 582 (6), 581 (14), 539 (2), 523 (3), 490 (3), 341 (5), 340(22), 301 (5), 300 (18), 299 (75), 283 (3), 282 (14), 256 (4), 242 (7),241 (5), 240 (16), 239 (62), 226 (6), 192 (3), 182 (8), 181 (5), 180(3), 168 (5), 166 (5), 154 (10), 131 (3), 106 (3), 95 (4), 94 (48), 93(5), 92 (8), 91 (100), 89 (5), 75 (6), 73 (8), 65 (3), 57 (3); IR (NEAT)3324 (br, NH), 3066, 3018, 2954, 2930, 2857, 1737, 1622, 1598, 1523,1489, 1464, 1409, 1363, 1327, 1230, 1205, 1168, 1115, 1080, 1030, 994,937, 839, 756, 697, 667, 638, 606, 472, 459, 443 cm⁻¹; exact mass calcdfor C₃₄H₄₆N₂O₈Si m/e 638.3024, obsd m/e 638.3223.

(2S)-N-[2-Allyloxycarbonylamino)-4-benzyloxy-5-methoxybenzoyl]-4-(acyloxy-2-ethyl)-2-(hydroxymethyl)-2,3-dihydropyrrole(29)

A solution of the silyl ether 28 (0.83 g, 1.30 mmol) in THF (14 mL) wastreated with H₂O (14 mL) and glacial acetic acid (42 mL). After 2 hoursstirring at room temperature TLC (50% EtOAc/Petroleum Ether) showed thecomplete consumption of starting material. The mixture was cooled (ice)and treated dropwise with a solution of NaHCO₃ (64 g) in H₂O (640 mL).The aqueous solution was extracted with EtOAc (3×100 mL) and thecombined organic layers were washed with H₂O (150 mL), brine (100 mL),dried (MgSO₄), filtered and concentrated in vacuo to give the crudeproduct as an orange oil. Purification by flash chromatography (60%EtOAc/Petroleum Ether) furnished the pure alcohol 29 as a white glass(0.537 g, 81%): [α]²¹ _(D)−83.60° (c=0.25, CHCl₃); ¹H NMR (270 MHz,CDCl₃) δ8.56 (br s, 1H), 7.89 (br s, 1H), 7.49-7.29 (m, 5H), 6.81 (s,1H), 6.28 (br s, 1H), 6.03-5.89 (m, 1H), 5.35 (ddd, 1H, J=17.22, 3.11,1.46, Hz), 5.25 (d, 1H, J=10.44 Hz), 5.19 (s, 2H), 4.80-4.70 (m, 1H),4.65-4.62 (m, 2H), 4.41-4.31 (m, 1H), 4.20-4.06 (m, 2H,), 3.84-3.77 (m,5H), 2.98-2.88 (m, 1H), 2.39 (t, 2H, J=6.51 Hz), 2.33-2.25 (m, 1H,),2.03 (s, 3H); ¹³C NMR (67.8 MHz, CDCl₃) δ170.8, 167.1, 153.5, 151.0,144.3, 136.1, 132.6, 132.4, 128.6, 128.1, 127.7, 126.3, 122.6, 118.1,112.2, 106.3, 70.7, 66.5, 65.8, 62.0, 61.7, 56.8, 35.4, 31.7, 27.8,20.9; MS (EI), m/z (relative intensity) 525 (M^(+.) +1, 5), 524 (M^(+.),14), 341 (5), 340 (16), 299 (2), 283 (3), 282 (14), 256 (4), 227 (5),208 (2), 192 (3), 190 (2), 186 (9), 185 (60), 168 (2), 167 (5), 166 (2),164 (2), 163 (2), 154 (3), 136 (3), 131 (3), 126 (7), 125 (53), 108 (2),107 (2), 106 (2), 105 (3), 95 (3), 94 (19), 93 (3), 92 (9), 91 (100), 83(2), 69 (2), 68 (3), 67 (3), 65 (5), 58 (6), 57 (17); IR (CHCl₃) 3335(br), 2933, 1732, 1599, 1524, 1455, 1434, 1408, 1231, 1170, 1112, 1029,995, 932, 868, 765, 698, 638, 606 cm⁻¹; exact mass calcd for C₂₈H₃₂N₂O,m/e 524.2159, obsd m/e 524.2074.

(11S,11aS)-2-(Acyloxy-2-ethyl)-10-allyloxycarbonyl-8-benzyloxy-11-hydroxy-7-methoxy-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(30)

Method A: A solution of DMSO (0.25 mL, 0.27 g, 3.49 mmol) in CH₂Cl₂ (10mL) was added dropwise over 35 minutes to a solution of oxalyl chloride(0.87 mL of a 2.0 M solution in CH₂Cl₂, 1.75 mmol) at −45° C.(liq.N₂/Chlorobenzene) under a nitrogen atmosphere. After stirring at−45° C. for 40 minutes, a solution of the alcohol 29 (0.51 g, 0.97 mmol)in CH₂Cl₂ (7 mL) was added dropwise over 35 minutes at −45° C. After 55minutes at −45° C., the mixture was treated dropwise with a solution ofTEA (0.57 mL, 0.41 g, 4.10 mmol) in CH₂Cl₂ (5 mL) over 40 minutes at−45° C. After a further 45 minutes, the reaction mixture was allowed towarm to room temperature and was diluted with CH₂Cl₂ (60 mL), washedwith 1N HCl (60 mL), H₂O (60 mL), brine (30 mL), dried (MgSO₄), filteredand evaporated in vacuo. TLC (80% EtOAc/Petroleum Ether) of the crudematerial revealed complete reaction. Purification by flashchromatography (50% EtOAc/Petroleum Ether) furnished the protectedcarbinolamine 30 as a creamy glass (0.25 g, 49%).

Method B: A solution of the alcohol 29 (0.21 g, 0.40 mmol) inCH₂Cl₂/CH₃CN (30 mL, 3:1) was treated with 4 Å powdered molecular sieves(0.15 g) and NMO (69 mg, 0.59 mmol). After 15 minutes stirring at roomtemperature, TPAP (6.9 mg, 19.8 μmol) was added and stirring continuedfor a further 1 hour at which point TLC (80% EtOAc/Petroleum Ether)showed product formation along with some unoxidised starting material.The mixture was then treated with a further quantity of NMO (35 mg, 0.30mmol) and TPAP (3.50 mg, 10 μmol), and allowed to stir for a further 1.5hours after which time TLC revealed complete reaction. The mixture wasevaporated in vacuo onto silica and subjected to flash chromatography(50% EtOAc/Petroleum Ether) to provide the protected carbinolamine 30 asa creamy glass (95 mg, 46%): [α]²⁰ _(D)=+113.85° (c=0.95, CHCl₃); ¹H NMR(270 MHz, CDCl₃) δ7.49-7.26 (m, 6H), 6.80 (s, 1H), 6.76 (s, 1H),5.79-5.59 (m, 1H), 5.75 (d, 1H, J=10.08 Hz), 5.19-5.05 (m, 4H),4.52-4.29 (m, 2H), 4.28-4.08 (m, 3H), 3.95-3.80 (m, 4H), 2.99 (dd, 1H,J=10.72, 16.94 Hz), 2.66 (d, 1H, J=16.86 Hz), 2.46 (t, 2H, J=6.41 Hz),2.06 (s, 3H); ¹³C NMR (67.8 MHz, CDCl₃) δ171.1, 163.1, 155.9, 150.3,149.1, 136.1, 131.8, 128.7, 128.6, 128.2, 127.3, 125.3, 124.4, 121.6,118.0, 114.8, 111.0, 85.9, 71.1, 66.8, 62.0, 70.7, 59.4, 56.2, 37.0,27.9, 21.0; MS (EI), m/z (relative intensity) 522 (M^(+.), 13), 463 (9),462 (13), 341 (8), 340 (32), 282 (11), 256 (3), 183 (5), 154 (3), 123(8), 94 (20), 91 (100), 65 (4), 57 (15); exact mass calcd for C₂₈H₃₀N₂O₈m/e 522.2002, obsd m/e 522.2008.

Example 1(c)

(11aS)-2-(Acyloxy-2-ethyl)-8-benzyloxy-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(31,UP2051, SJG-301)

A catalytic amount of tetrakis(triphenylphosphine)palladium (5.26 mg,4.55 μmol) was added to a stirred solution of the Alloc-protectedcarbinolamine 30 (95 mg, 0.18 mmol), triphenylphosphine (2.39 mg,9.10,mol) and pyrrolidine (13.6 mg, 0.19 mmol) in CH₂Cl₂ (10 mL). After1 hour stirring at room temperature under a nitrogen atmosphere, TLC(97% CHCl₃/MeOH) revealed the complete consumption of starting material.The solvent was evaporated in vacuo and the crude residue was purifiedby flash chromatography (99.5% CHCl₃/MeOH) to afford the PBD (31,SJG-301, UP2051) as an orange glass which was repeatedly evaporated invacuo with CHCl₃ in order to provide the N10-C11 imine form (66.3 mg,87%): [α]²¹ _(D)=+741.67° (c=0.66, CHCl₃); ¹H NMR (270 MHz, CDCl₃)(imine) δ7.78 (d, 1H, J=4.03 Hz), 7.70-7.28 (m, 6H), 6.83 (s, 1H), 6.82(s, 1H), 5.19-5.18 (m, 2H), 4.27-4.16 (m, 2H), 3.94 (s, 3H), 3.44-3.35(m, 1H), 3.28-3.15 (m, 1H), 3.04-2.97 (m, 1H), 2.52-2.47 (m, 2H), 2.06(s, 3H); ¹³C NMR (67.8 MHz, CDCl₃) (Rotamers) δ170.9, 162.6, 161.1,150.9, 148.2, 140.1, 136.1, 132.1, 132.0, 128.7, 128.6, 128.1, 127.3,124.7, 121.4, 111.9, 111.6, 70.8, 61.9, 56.2, 53.6, 37.4, 27.9, 21.0; MS(EI), m/z (relative intensity) 421 (M^(+.) +1, 4), 420 (M^(+.), 14), 419(12), 418 (36), 361 (6), 360 (20), 328 (3), 313 (8), 270 (4), 269 (7),268 (9), 267 (22), 256 (4), 129 (3), 105 (3), 94 (4), 93 (3), 92 (12),91 (100), 83 (3), 80 (3), 73 (5), 71 (3), 69 (3), 65 (5), 60 (4), 57(5), 55 (4); IR (CHCl₃) 3313 (br), 2957, 2934, 1736, 1598, 1509, 1455,1437, 1384, 1243, 1179, 1120, 1096, 1037, 753, 696, 666, 542 cm⁻¹; exactmass calcd for in C₂₄H₂₄N₂O₅ m/e 420.1685, obsd m/e 420.1750.

(11s,11aS)-10-Allyloxycarbonyl-8-benzyloxy-11-hydroxy-2-(hydroxy-2-ethyl)-7-methoxy-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(32)

A solution of K₂CO₃ (328 mg, 2.38 mmol) in H₂O (6 mL) was added dropwiseto a stirred solution of the acyl compound 30 (0.248 g, 0.475 mmol) inCH₂CL₂ (3 mL) and MeOH (8 mL). After stirring for 16 hours at roomtemperature TLC (EtOAc) revealed complete reaction. The MeOH/CH₂Cl₂ wasevaporated in vacuo to give a cloudy aqueous solution which was dilutedwith H₂O (30 mL) and extracted with EtOAc (3×30 mL). The combinedorganic layers were then washed with brine (30 mL), dried (MgSO₄),filtered and evaporated in vacuo to provide a creamy oil. Purificationby flash chromatography (97% CHCl₃/MeOH) furnished the homoallylicalcohol 32 as a transparent colourless glass (178 mg, 78%): [α]²¹_(D)+48.43° (c=1.56, CHCl₃); ¹H NMR (270 MHz, CDCl₃) δ7.43-7.24 (m, 6H),6.84 (s, 1H), 6.73 (s, 1H), 5.74-5.55 (m, 1H), 5.73 (d, 1H, J=8.79 Hz),5.19-5.06 (m, 4H), 4.46-4.23 (m, 2H), 3.92-3.70 (m, 6H), 3.07-2.97 (m,1H), 2.67 (d, 1H, J=16.49 Hz), 2.40-2.17 (m, 2H); ¹³C NMR (67.8 MHz,CDCl₃) δ163.1, 155.8, 150.3, 149.1, 136.1, 131.8, 128.6, 128.1, 127.7,127.4, 125.3, 124.1, 124.0, 123.1, 123.0, 117.9, 114.9, 110.9, 86.0,71.1, 66.7, 60.3, 59.6, 56.2, 37.1, 31.5; MS (EI), m/z (relativeintensity) 482 (M^(+.) +2, 4), 481 (M^(+.) +1, 10), 480 (M^(+.), 26),449 (4), 378 (12), 347 (7), 341 (7), 340 (25), 339 (4), 284 (4), 282(10), 143 (4), 141 (13), 131 (6), 112 (24), 110 (4), 94 (10), 92 (9), 91(100), 80 (4), 70 (5), 69 (7), 65 (4), 58 (11), 57 (29); exact masscalcd for C₂₆H₂₈N₂O₇ m/e 480.1897, obsd m/e 480.1886.

Example 1(d)

(11aS)-8-Benzyloxy-2-(hydroxy-2-ethyl)-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(33, UP2052, SJG-303)

A catalytic amount of tetrakis(triphenylphosphine)palladium (9.39 mg,8.13 μmol) was added to a stirred solution of the Alloc-protectedcarbinolamine 30 (156 mg, 0.33 mmol), triphenylphosphine (4.26 mg, 16.3μmol) and pyrrolidine (24.3 mg, 0.34 mmol) in CH₂Cl₂ (15 mL). After 1hour 50 minutesutes stirring at room temperature under a nitrogenatmosphere, TLC (90% CHCl₃/MeOH) revealed the complete consumption ofstarting material. The solvent was evaporated in vacuo and the cruderesidue was purified by flash chromatography (98% CHCl₃/MeOH) to affordthe PBD (33, SJG-303, UP2052) as an orange glass which was repeatedlyevaporated in vacuo with CHCl₃ in order to provide the N10-C11 imineform (103 mg, 84%): ¹H NMR (270 MHz, CDCl₃) (Rotamers) δ7.75 (d, 1H,J=4.03 Hz), 7.58-7.22 (m, 6H), 6.82-6.80 (m, 2H), 5.17-4.88 (m, 2H),4.65-4.20 (m, 2H), 3.91 (s, 3H), 3.35-3.25 (m, 1H), 3.18-3.15 (m, 1H),3.04-2.97 (m, 1H), 2.52-2.47 (m, 2H); ¹³C NMR (67.8 MHz, CDCl₃)(Rotamers) δ162.8, 161.1, 152.3, 150.9, 148.1, 142.3, 138.3, 136.4,128.7, 128.6, 128.2, 127.4, 124.2, 123.1, 111.8, 111.6, 70.8, 60.4,56.2, 53.6, 37.7, 31.5; MS (EI), m/z (relative intensity) 380 (13), 379(11), 378 (M^(+.), 42), 377 (36), 376 (77), 375 (6), 347 (8), 345 (5),334 (5), 333 (19), 288 (14), 287 (14), 286 (36), 285 (50), 272 (6), 271(22), 269 (6), 268 (6), 267 (5), 259 (5), 257 (13), 255 (24), 243 (15),155 (6), 136 (5), 124 (7), 106 (6), 93 (6), 92 (38), 91 (100), 65 (16),63 (5), 51 (5); IR (CHCl₃) 3313, 2918, 1623, 1598, 1568, 1509, 1455,1436, 1386, 1328, 1243, 1218, 1175, 1130 1061, 1007, 870, 831, 792, 752,697, 662 cm⁻¹; exact mass calculated for C₂₂H₂₂N₂O₄ m/e 378.1580, obsdm/e 378.1576.

Repeated evaporation in vacuo of UP2052 with CH₃OH provided the N10-C11methyl ether forms: ¹H NMR (270 MHz, CDCl₃) (Rotamers) δ7.66-7.22 (m,6H), 6.82-6.81 (m, 2H), 5.21-4.76 (m, 2H), 4.61-4.15 (m, 1H), 4.03-3.71(m, 5H), 3.44 (s, 3H), 3.35-1.92 (m, 7H).

Example 1(e)

Synthesis of the C7,C8-Dimethoxy-C2-Methoxycarbonylmethyl PBD AN-SJG(42, UP2065) (See FIG. 4)

(2S)(4R)-N-(4,5-Dimethoxy-2-nitrobenzoyl)-2-(tert-butyldimethylsilyloxymethyl)-4-hydroxypyrrolidine(35)

A catalytic amount of DMF (2 drops) was added to a stirred solution ofthe nitro-acid 34 (12.45 g, 54.8 mmol) and oxalyl chloride (5.75 mL,8.37 g, 65.9 mmol) in CH₂Cl₂ (300 mL). After 16 hours at roomtemperature the resulting acid chloride solution was added dropwise over4.5 hours to a stirred mixture of the amine 2 (12.65 g, 54.8 mmol) andTEA (13.86 g, 19.1 mL, 137 mmol) in CH₂Cl₂ (300 mL) at 0° C.(ice/acetone) under a nitrogen atmosphere. The reaction mixture wasallowed to warm to room temperature and stirred for a further 2.5 h. Themixture was washed with saturated NaHCO₃ (300 mL), saturated NH₄Cl (300mL), H₂O (250 mL), brine (300 mL), dried (MgSO₄), filtered andevaporated in vacuo to give the crude product as a dark orange oil.Purification by flash chromatography (80% EtOAc/Petroleum Ether)isolated the pure amide 35 as a sticky orange oil (18.11 g, 75%): [α]²²_(D)=−105.7° (c=1.17, CHCl₃); ¹H NMR (270 MHz, CDCl₃) (Rotamers) δ7.71and 7.68 (s×2, 1H), 6.86 and 6.79 (s×2, 1H), 4.50 and 4.38 (br s×2, 2H),4.13-4.10 (m, 1H), 3.98 (s, 3H), 3.94 (s, 3H), 3.78-3.74 (m, 1H),3.35-3.27 (m, 1H), 3.07 (d, 1H, J=11.17 Hz), 3.01-2.79 (br s, 1H),2.35-2.26 (m, 1H), 2.11-2.04 (m, 1H), 0.91 and 0.81 (s×2, 9H), 0.10,0.09, −0.07, and −0.10 (s×4, 6H); ¹³C NMR (67.8 MHz, CDCl₃) (Rotamers)δ166.6, 154.2 and 154.1, 149.3 and 148.9, 137.5, 128.0, 109.2, 107.1,70.1 and 69.4, 64.7 and 62.5, 59.0 and 54.9, 57.3, 56.6, 56.5, 37.4 and36.3, 25.9 and 25.7, 18.2, −5.4, −5.5 and −5.7; MS (EI), m/z (relativeintensity) 440 (M^(+.), 2), 426 (9), 386 (4), 385 (20), 384 (65), 383(100), 367 (4), 320 (4), 308 (7), 295 (8), 286 (5), 211 (15), 210 (100),194 (12), 180 (4), 165 (17), 164 (8), 137 (4), 136 (25), 121 (4), 93(6), 91 (9), 82 (6), 75 (15), 73 (15), 59 (4), 57 (4); IR (NEAT) 3391(br, OH), 3012, 2952, 2931, 2857, 1616, 1578, 1522, 1456, 1436, 1388,1338, 1279, 1225, 1183, 1151, 1074, 1053, 1029, 1004, 939, 870, 836,816, 785, 757, 668, 650, 620 cm⁻¹; exact mass calcd for C₂₀H₃₂N₂O₇Si m/e440.1979, obsd m/e 440.1903.

(2S)(4R)-N-(2-Amino-4,5-dimethoxybenzoyl)-2-(tert-butyldimethylsilyloxymethyl)-4-hydroxypyrrolidine(36)

A solution of hydrazine (6.59 g, 6.40 mL, 205.5 mmol) in MeOH (110 mL)was added dropwise to a solution of the nitro-compound 35 (18.1 g, 41.1mmol), over anti-bumping granules and Raney Ni (2.6 g) in MeOH (325 mL)and heated at reflux. After 1 hour at reflux TLC (95% CHCl₃/MeOH)revealed some amine formation. The reaction mixture was treated withfurther Raney Ni (2.6 g) and hydrazine (6.40 mL) in MeOH (50 mL) and washeated at reflux for an additional 30 minutes at which point TLCrevealed reaction completion. The reaction mixture was then treated withsufficient Raney Ni to decompose any remaining hydrazine and heated atreflux for a further 1.5 h. Following cooling to room temperature themixture was filtered through a sinter and the resulting filtrateevaporated in vacuo. The resulting residue was then treated with CH₂Cl₂(300 mL), dried (MgSO₄), filtered and evaporated in vacuo to provide theamine 36 as a green oil (16.03 g, 95%): [α]²² _(D)=−116.32° (c=0.31,CHCl₃); ¹H NMR (270 MHz, CDCl₃) (Rotamers) δ6.70 (s, 1H), 6.28 (s, 1H),4.51-4.49 (m, 1H), 4.36-4.34 (m, 1H), 4.06-3.77 (m, 10H), 3.61-3.50 (m,3H), 2.23-2.21 (m, 1H), 2.01-1.98 (m, 1H), 0.89 (s, 9H), 0.04 (s, 6H);¹³C NMR (67.8 MHz, CDCl₃) (Rotamers) δ170.2, 151.5, 141.2, 140.5, 112.2,112.0, 101.1, 70.4, 62.6, 59.0, 56.9, 56.6, 55.8, 35.7, 25.9 and 25.7,18.2, −5.4 and −5.5; MS (EI), m/z (relative intensity) 412 (M^(+.) +2,3), 411 (M^(+.) +1, 10), 410 (M^(+.), 32), 354 (6), 353 (23), 263 (3),212 (5), 181 (11), 180 (100), 179 (3), 165 (3), 164 (6), 152 (10), 137(4), 136 (4), 125 (5), 120 (3), 100 (3), 94 (6), 75 (9), 73 (7), 57 (3);IR (CHCl₃) 3353 (br), 2953, 2930, 2857, 1623, 1594, 1558, 1517, 1464,1435, 1404, 1260, 1234, 1215, 1175, 1119, 1060, 1005, 915, 836, 777,755, 666 cm⁻¹; exact mass calcd for C₂₀H₃₄N₂O₅Si m/e 410.2237, obsd m/e410.2281.

(2S)(4R)-N-[(2-Allyloxycarbonylamino)-4,5-dimethoxybenzoyl]-2-(tert-butyldimethylsilyloxymethyl)-4-hydroxypyrrolidine(37)

A solution of the amine 36 (16.03 g, 39 mmol) in CH₂Cl₂ (450 mL) wascooled to 0° C. (ice/acetone) and treated with pyridine (6.94 mL, 6.78g, 85.8 mmol). A solution of allyl chloroformate (4.35 mL, 4.94 g, 40.95mmol) in CH₂Cl₂ (90 mL) was then added dropwise to the stirred mixture.The reaction mixture was allowed to warm to room temperature and stirredfor a further 1.5 h, at which point TLC (EtOAc) revealed completeconsumption of amine 36. The reaction mixture was washed with saturatedCuSO₄ (300 mL), H₂O (300 mL), brine (300 mL), dried (MgSO₄), filteredand evaporated in vacuo. The crude residue was purified by flashchromatography (35% EtOAc/Petroleum Ether) to afford the purealloc-amino compound 37 as a clear oil (16.78 g, 87%): [α]²³_(D)=−93.35° (c=0.27, CHCl₃); ¹H NMR (270 MHz, CDCl₃) (Rotamers) δ8.93(br s, 1H), 7.72 (S, 1H), 6.77 (S, 1H), 6.01-5.87 (m, 1H), 5.34 (dd, 1H,J=17.22, 3.12 Hz), 5.23 (dd, 1H, J=10.44, 1.29 Hz), 4.63-4.55 (m, 3H),4.40-4.38 (m, 1H), 4.15-4.08 (m, 1H), 3.91 (S, 3H), 3.81 (s, 3H),3.62-3.55 (m, 3H), 2.34-2.24 (m, 2H), 2.07-1.99 (m, 1H), 0.89 (s, 9H),0.05 and 0.04 (s×2, 6H); ¹³C NMR (67.8 MHz, CDCl₃) (Rotamers) δ169.5,153.8, 150.9, 143.8, 132.5, 118.0, 115.9, 111.0, 104.6, 70.5, 65.8,62.2, 59.0, 57.2, 56.2, 56.0, 35.7 and 31.1, 25.8, 18.1, −5.4 and −5.5;MS (EI), m/z (relative intensity) 496 (M^(+.) +2, 6), 495 (M^(+.) +1,18), 494 (M^(+.), 50), 439 (11), 438 (29), 437 (100), 380 (4), 379 (14),337 (13), 336(4), 265 (15), 264 (91), 263 (4), 258 (6), 224 (4), 223(15), 220 (11), 212 (7), 208 (4), 207 (11), 206 (75), 192 (5), 180 (20),179 (18), 174 (15), 172 (4), 164 (7), 156 (5), 152 (5), 150 (6), 136(4), 99 (9), 86 (16), 75 (10), 73 (11), 57 (6); IR (CHCl₃) 3337 (br),2952, 2930, 2857, 1733, 1600, 1522, 1458, 1420, 1399, 1327, 1288, 1261,1229, 1203, 1165, 1121, 1039, 1004, 931, 836, 777, 668 cm⁻¹; exact masscalcd for C₂₄H₃₈N₂O₇Si m/e 494.2448, obsd m/e 494.2365.

(2S)-N-[(2-Allyloxycarbonylamino)-4,5-dimethoxybenzoyl]-2-(tert-butyldimethylsilyloxymethyl)-4-oxopyrrolidine(38)

A solution of DMSO (7.24 mL, 7.97 g, 102 mmol) in CH₂Cl₂ (150 mL) wasadded dropwise over 2 hours to a solution of oxalyl chloride (25.5 mL ofa 2.0 M solution in CH₂Cl₂, 51.0 mmol) at −60° C. (liq.N₂/CHCl₃) under anitrogen atmosphere. After stirring at −50° C. for 1 hour, a solution ofthe alcohol 37 (16.75 g, 33.9 mmol) in CH₂Cl₂ (250 mL) was addeddropwise over a period of 2 h. After 1 hour at −55° C., the mixture wastreated dropwise with a solution of TEA (32.2 mL, 23.4 g, 231 mmol) inCH₂Cl₂ (100 mL) and allowed to warm to room temperature. The reactionmixture was treated with brine (250 mL) and washed with cold 1N HCl(2×300 mL). TLC (50% EtOAc/Petroleum Ether) analysis of the CH₂OC₂ layerrevealed complete reaction. The layers were separated and the organicphase washed with H₂O (300 mL), brine (300 mL), dried (MgSO₄), filteredand concentrated in vacuo to give the ketone 38 as an orange glass(16.37 g, 98%): [α]²¹ _(D)=−9.96° (c=1.51, CHCl₃); ¹H NMR (270 MHz,CDCl₃) δ8.69 (s, 1H), 7.82 (s, 1H), 6.75 (s, 1H), 6.01-5.89 (m, 1H),5.36 (dd, 1H, J=17.22, 3.11 Hz), 5.28-5.23 (m, 1H), 5.20-4.95 (m, 1H),4.65-4.62 (m, 2H), 4.20-3.83 (m, 9H), 3.67-3.56 (m, 1H), 2.74 (dd, 1H,J=17.86, 9.44 Hz), 2.52 (d, 1H, J=17.95 Hz), 0.87 (s, 9H), 0.05 (s, 6H);¹³C NMR (67.8 MHz, CDCl₃) (Rotamers) δ208.9, 169.1, 153.5, 151.3, 143.9,132.4, 118.2, 114.4, 110.1, 104.6, 66.1, 65.8, 56.2, 56.0, 39.7, 25.6,18.0, −5.7 and −5.8; MS (EI), m/z (relative intensity) 494 (M^(+.) +2,6), 493 (M^(+.) +1, 16), 492 (M^(+.), 43), 437 (8), 436 (22), 435 (74),377 (11), 336 (6), 335 (21), 334 (8), 294 (8), 265 (9), 264 (50), 250(5), 223 (17), 220 (18), 208 (7), 207 (15), 206 (100), 192 (9), 180(23), 179 (28), 172 (33), 171 (10), 164 (16), 155 (7), 152 (9), 150(16), 136 (13), 115 (14), 108 (6), 88 (6), 75 (20), 73 (33), 59 (13), 58(6), 57 (62), 56 (14); IR (NEAT) 3337 (br, NH), 3086, 3019, 2954, 2932,2858, 1766, 1732, 1623, 1603, 1520, 1464, 1398, 1362, 1332, 1313, 1287,1262, 1204, 1166, 1110, 1052, 1038, 1004, 938, 870, 838, 810, 756, 666,621, 600 cm⁻¹; exact mass calcd for C₂₄H₃₆N₂O₇Si m/e 492.2292, obsd m/e492.2349.

2S)-N-[(2-Allyloxycarbonylamino)-4,5-dimethoxybenzoyl]-2-(tert-butyldimethylsilyloxymethyl)-4-(methoxycarbonylmethyl)-2,3-dihydropyrrole(39)

Petroleum ether (70 mL) was added to a sample of NaH (0.41 g of a 60%dispersion in oil, 10.16 mmol) and stirred at room temperature under anitrogen atmosphere. After 0.5 hours the mixture was allowed to settleand the Petroleum Ether was transferred from the flask via adouble-tipped needle under nitrogen. THF (60 mL) was added to theremaining residue and the mixture was cooled to 0° C. (ice/acetone). Thecool solution was treated dropwise with a solution ofmethyldiethylphosphonoacetate (1.86 mL, 2.14 g, 10.16 mmol) in THF (60mL) under nitrogen. After 1.5 hours at room temperature, the mixture wascooled to 0° C. and treated dropwise with a solution of the ketone 38(2.0 g, 4.07 mmol) in THF (36 mL) under nitrogen. After 16 hours at roomtemperature, TLC (20% EtOAc/Petroleum Ether) revealed reactioncompletion. The THF was evaporated in vacuo and the mixture partitionedbetween saturated NaHCO₃ (100 mL) and EtOAc (100 mL). The layers wereseparated and the aqueous layer extracted with EtOAc (2×100 mL). Thecombined organic layers were washed with H₂O (100 mL), brine (100 mL),dried (MgSO₄), filtered and concentrated in vacuo to give a dark redoil. Purification by flash chromatography (15% EtOAc/Petroleum Ether)furnished the endo-ester 39 as a golden oil (1.63 g, 73%): ¹H NMR (270MHz, CDCl₃) (Rotamers) δ8.82 (br s, 1H), 7.86 (s, 1H), 6.79 (s, 1H),6.46 (br s, 1H), 6.03-5.89 (m, 1H), 5.39-5.32 (m, 1H), 5.24 (dd, 1H,J=10.44, 1.28 Hz), 4.70-4.59 (m, 3H), 3.99-3.61 (m, 11H), 3.08 (s, 2H),2.91-2.82 (m, 1H), 2.75-2.66 (m, 1H), 0.92-0.79 (m, 9H), 0.12—0.03 (m,6H); ¹³C NMR (67.8 MHz, CDCl₃) (Rotamers) δ170.7, 165.8, 153.5, 151.3,143.7, 132.8, 132.5, 128.2, 118.1, 118.0, 117.9, 111.3, 104.3, 65.7,62.3, 59.5 and 59.4, 56.4, 56.0, 52.0, 34.7, 33.9, 25.8, 18.1, −5.4; MS(EI), m/z (relative intensity) 549 (M^(+.) +1, 7), 548 (M^(+.), 17), 525(13), 507 (14), 492 (6), 491 (18), 489 (8), 449 (7), 347 (11), 287 (6),286 (20), 285 (82), 265 (10), 264 (51), 263 (9), 244 (9), 242 (7), 228(19), 227 (8), 226 (18), 224 (6), 223 (22), 220 (12), 208 (6), 207 (18),206 (100), 192 (7), 180 (18), 179 (21), 168 (16), 164 (10),152 (13), 150(8), 141 (8), 140 (73), 139 (13), 136 (6), 108 (6), 89 (9), 80 (15), 75(15), 73 (19), 57 (6); exact mass calcd for C₂₇H₄₀N₂O₈Si m/e 548.2554,obsd m/e 548.2560.

(2S)-N-[(2-Allyloxycarbonylamino)-4,5-dimethoxybenzoyl]-2-(hydroxymethyl)-4-(methoxycarbonylmethyl)-2,3-dihydropyrrole(40)

A solution of the silyl ether 39 (1.63 g, 2.97 mmol) in THF (12.6 mL)was treated with H₂O (12.6 mL) and glacial acetic acid (38 mL). After 2hours stirring at room temperature TLC (60% EtOAc/Petroleum Ether)showed the complete consumption of starting material. The mixture wascooled (ice) and treated dropwise with a solution of NaHCO₃ (61.6 g) inH₂O (616 mL). The aqueous solution was extracted with EtOAc (3×150 mL)and the combined organic layers were washed with H₂O (150 mL), brine(100 mL), dried (MgSO₄), filtered and concentrated in vacuo to give thecrude alcohol 40 as an orange oil (1.27 g, 98%): MS (EI), m/z (relativeintensity) 435 (M^(+.) +1, 6), 434 (M^(+.), 23), 347 (5), 317 (4), 281(6), 265 (8), 264 (44), 263 (8), 224 (5), 223 (24), 222 (5), 220 (9) 207(15), 206 (94), 192 (5), 180 (18), 179 (18), 172 (12), 171 (100), 164(12), 152 (7), 150 (7), 141 (6), 140 (53), 136 (9), 112 (11), 108 (6),80 (12), 69 (7); exact mass calcd for C₂₁H₂₆N₂O₈ m/e 434.1689, obsd m/e434.1606.

(11S,11aS)-10-Allyloxycarbonyl-7,8-dimethoxy-11-hydroxy-2-(methoxycarbonylmethyl)-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(41)

A solution of DMSO (0.75 mL, 0.82 g, 10.5 mmol) in CH₂Cl₂ (22 mL) wasadded dropwise over 1 hour 20 minutes to a solution of oxalyl chloride(2.63 mL of a 2.0 M solution in CH₂Cl₂, 5.26 mmol) at −45° C.(liq.N₂/Chlorobenzene) under a nitrogen atmosphere. After stirring at−45° C. for 1 h, a solution of the alcohol 40 (1.27 g, 2.92 mmol) inCH₂Cl₂ (22 mL) was added dropwise over 1 hour at −45° C. After 50minutes at −45° C., the mixture was treated dropwise with a solution ofTEA (1.71 mL, 1.24 g, 12.29 mmol) in CH₂Cl₂ (11 mL) over 30 minutes at−45° C. After a further 30 minutes, the reaction mixture was allowed towarm to room temperature and was diluted with CH₂Cl₂ (20 mL), washedwith 1N HCl (100 mL), H₂O (100 mL), brine (100 mL), dried (MgSO₄),filtered and evaporated in vacuo. TLC (80% EtOAc/Petroleum Ether) of thecrude material revealed reaction completion. Purification by flashchromatography (55% EtOAc/Petroleum Ether) furnished the protectedcarbinolamine 41 as a white glass (0.68 g, 54%): [α]²² _(D)=+219.78°(c=0.12, CHCl,); ¹H NMR (270 MHz, CDCl₃) δ7.23 (s, 1H), 6.91 (s, 1H),6.70 (s, 1H), 5.90-5.80 (m, 2H), 5.17-5.13 (m, 2H), 4.70 (dd, 1H,J=13.37, 5.31 Hz), 4.50-4.43 (m, 1H), 3.98-3.75 (m, 8H), 3.71 (s, 3H),3.20-3.05 (m, 3H), 2.75 (d, 1H, J=17.04 Hz); ¹³C NMR (67.8 MHz, CDCl₃)δ170.7, 163.3, 155.9, 151.1, 148.5, 131.7, 128.3, 126.2, 124.7, 118.1,117.6, 112.6, 110.6, 86.0, 66.8, 59.4, 56.2, 52.1, 37.0, 33.7; MS (EI),m/z (relative intensity) 434 (M^(+.) +2, 6), 433 (M^(+.) +1, 21), 432(M^(+.), 74), 414 (8), 373 (14), 329 (7), 293 (20), 292 (20), 265 (19),264 (100), 263 (33), 248 (25), 224 (6), 223 (25), 220 (14), 209 (8), 208(52), 207 (24), 206 (92), 192 (15), 191 (6), 190 (7), 180 (18), 179(23), 169 (23), 165 (10), 164 (17), 152 (12), 150 (14), 149 (8), 141(9), 140 (60), 136 (11), 125 (6), 120 (5), 110 (8), 108 (15), 81 (9), 80(45), 57 (7); IR (CHCl₃) 3385 (br), 2918, 2849, 1707, 1625, 1605, 1516,1457, 1436, 1405, 1311, 1282, 1245, 1217, 1172, 1116, 1046, 1001, 968,933, 874, 855, 666 cm⁻¹.

(11aS)-7,8-Dimethoxy-2-(methoxycarbonylmethyl)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(42, UP2065, AN-SJG)

A catalytic amount of tetrakis(triphenylphosphine)palladium (44.0 mg,38.0 μmol) was added to a stirred solution of the Alloc-protectedcarbinolamine 41 (0.66 g, 1.53 mmol), triphenylphosphine (20.0 mg, 77.0μmol) and pyrrolidine (114 mg, 1.60 mmol) in CH₂Cl₂ (100 mL). After 2hours stirring at room temperature under a nitrogen atmosphere, TLC (99%CHCl₃/MeOH) revealed the complete consumption of starting material. Thesolvent was evaporated in vacuo and the crude residue was purified byflash chromatography (98% CHCl₃/MeOH) to afford the PBD (42, AN-SJG,UP2065) as an orange glass which was repeatedly evaporated in vacuo withCHCl₃ in order to provide the N10-C11 imine form (481 mg, 95%): [α]²²_(D)=+401.84° (c=1.00, CHCl₃); ¹H NMR (270 MHz, CDCl₃) δ7.87-7.85 (m,1H), 7.49 (s, 1H), 6.93 (s, 1H), 6.81 (s, 1H), 4.34-4.27 (m, 1H), 3.95(s, 3H), 3.93 (s, 3H), 3.74 (s, 3H), 3.34 (d, 1H, J=16.85 Hz), 3.24 (s,2H), 3.19-3.10 (m, 1H); ¹³C NMR (67.8 MHz, CDCl₃) δ170.6, 162.7, 161.4,151.8, 147.7, 140.4, 126.5, 119.0, 117.4, 111.5, 109.8, 56.2, 56.1,53.8, 52.1, 37.4, 33.6; MS (EI), m/z (relative intensity) 332 (^(+.) +2,5), 331 (M^(+.) +1, 9), 330 (M^(+.), 41), 329 (28), 328 (100), 313 (18),272 (8), 271 (24), 270 (14), 269 (27), 262 (7), 257 (12), 255 (5), 242(6), 225 (7), 197 (4), 192 (16), 191 (16), 183 (6), 164 (14), 136 (11),135 (9), 106 (9), 80 (17), 53 (5); IR (CHCl₃) 3329 (br), 3112, 2952,2842, 1737, 1626, 1602, 1512, 1453, 1436, 1381, 1356, 1246, 1213, 1173,1096, 1069, 1008, 875, 840, 786, 666, 620, 574, 537 cm⁻¹; exact masscalcd for C₁₇H₁₈N₂O, m/e 330.1216, obsd m/e 330.1237.

Example 1(f)

Synthesis of KEC-570 (56, UP-2053) (See FIG. 5 a/5 b)

1′,3′-Bis(4-carboxy-2-methoxyphenoxy)propane (43)

A solution of diiodopropane (8.79 g, 29.7 mmol) in THF (50 mL), wasadded dropwise over a period of 4 hours to a vigorously stirred solutionof vanillic acid (10 g, 59.5 mmol) in THF (100 mL) and aqueous NaOH (225mL, 0.5 M) at 65° C. in the absence of light (foil-wrapped flask). Afterheating at reflux for 48 hours in the dark, the suspension was cooled,washed with hexane (3×100 mL) and the THF removed by evaporation invacuo. The aqueous residue was acidified to pH 1 with conc. HCl and theresultant precipitate collected by filtration, dried and recrystallisedfrom glacial acetic acid to afford the corresponding bis-carboxylic acid(143) as a white crystalline solid (9.4 g, 84%). mp 238-240° C.; ¹H-NMR(DMSO-d₆): δ2.23 (t, 2H, J=6.0 Hz, H13), 3.80 (s, 6H, CH₃O), 4.20 (t,4H, J=6.0 Hz, H12), 7.09 (d, 2H, J=8.4 Hz, H10), 7.45 (d, 2H, J=1.8 Hz,H6) 7.54 (dd, 2H, J=8.4 Hz, 1.8 Hz, H9), 12.76 (bs, 2H, CO₂H); ¹³C-NMR(DMSO-d₆) δ28.4 (C13), 55.4 (CH₃O), 64.8 (C12), 111.9 (C9), 112.0 (C6),122.9 (C1O), 123.0 (Q), 148.3 (Q), 151.6 (Q), 167.0 (C═O). IR (KBr):ν=3600-2000, 1680 (C═O), 1600 (C═C), 1515, 1465, 1430, 1345, 1310, 1270,1225 (C—O—C), 1180, 1140, 1115, 1030, 990, 970, 950, 925, 875, 850, 825,765, 725, 645 cm⁻¹. MS (EI): m/z (relative intensity) 376 (M^(+.), 28),360 (3), 249 (2), 209 (45), 165 (29), 153 (16), 151 (19), 137 (19), 121(7), 78 (15), 44 (100); HRMS: Calcd for C₁₉H₂₀O₈=376.1158 found376.1168.

1′,3′-Bis(4-carboxy-2-methoxy-5-nitrophenoxy)propane (44)

The diacid 43 (2.0 g, 5.30 mmol) was added portionwise to conc. HNO₃ (40mL) at −10° C. and stirred to room temperature over 12 h. The reactionmixture was poured on to ice (400 mL) and the resulting precipitatecollected by filtration, washed with ether (3×50 mL) and dried to affordthe nitro acid (121) as a yellow solid (1.73 g, 70%). m.p. 243-2460C.¹H-NMR (DMSO-d₆): δ2.25 (t, 2H, J=5.9 Hz, H13), 3.90 (s, 6H, CH₃O), 4.27(t, 4H, J=5.9 Hz, H12), 7.29 (s, 2H, H6), 7.62 (s, 2H, H9), 13.6 (bs,2H, CO₂H). ^(—)C-NMR (DMSO-d₆) δ28.0 (C13), 56.3 (CH₃O), 65.7 (C12),108.0 (C9), 111.2 (C6), 121.1 (C5), 141.3(Q), 149.1 (C8), 151.7 (O),165.9 (C═O). IR (KBr): ν=3620-2280, 1700 (C═O), 1595 (C═C), 1570, 1515(NO₂), 1460, 1415, 1350 (NO₂), 1270, 1210, 1180, 1135, 1045, 930, 880,810, 750, 730, 645 cm⁻¹. MS (EI): m/z (relative intensity) 467 (MH^(+.),1), 450 (1), 436 (3), 423 (8), 378 (4), 268 (1), 255 (4), 236 (4), 210(7), 194 (2), 182 (7), 164 (14), 153 (2), 123 (3), 91 (6), 77 (3), 55(5), 44 (100). HRMS (EI) m/z calcd for C₁₉H₁₈N₂O₁₂=466.0860 found466.0871.

(2S,4R)-N-(Benzoxycarbonyl)-2-carboxy-4-hydroxypyrrolidine (45)

A solution of benzyl chloroformate (12.5 mL, 87.7 mL) in toluene (40 mL)was added to a solution of trans-4-hydroxy-L-proline 11 (10 g, 76.3mmol) and NaHCO₃ (16 g, 190 mmol) in H₂O (165 mL) over a period of 15minutes. After stirring at room temperature for 12 hours the two phaseswere allowed to separate. The aqueous phase was washed with diethylether (4×50 mL), cooled in an ice bath, and then acidified to pH 2 withconc. HCl. The resultant product was extracted with ethyl acetate (5×50mL) and the combined organic extracts were dried (MgSO₄) and the excesssolvent evaporated in vacuo to afford a colourless viscous oil (20.30 g,100%). [α]²⁷ _(D)=−565° (c 0.1, MeOH). ¹H NMR (CDCl₃): δ2.07-2.31 (m,3H, H1), 3.52-3.59 (m, 2H, H3), 4.43-4.53 (m, 2H, H2, H11a), 5.8 and5.11 (s, 2H, minor and major rotamers of H6, 1:2), 6.0 (bs, 2H, OH),7.26-7.29 and 7.32-7.34 (m, 5H, minor and major rotamers of H arom,1:2). IR (thin film): ν=3414 (OH), 2940 (OH), 1682 (C═O), 1495, 1429,1359 (CO₂−), 1314, 1269, 1205, 1180, 1174, 1127, 1082, 1051, 993, 914,866, 826, 769, 741, 697 cm⁻¹. MS (EI): m/e (relative intensity): 266(M^(+.), 1), 265 (6), 220 (5), 176 (15), 130 (34), 108 (2). 91 (100), 86(4), 68 (11). HRMS calcd. for C₁₃H₁₅NO₅=265.0950 found 265.0976.

(2S,4R)-N-(Benzoxycarbonyl)-2-methyoxycarbonyl-4-hydroxyproline (46)

A solution of (2S,4R)-N-(Benzoxycarbonyl)-2-carboxy-4-hydroxypyrrolidine(45) (20.30 g, 76.3 mmol) in dry methanol (300 mL) was heated at refluxfor 18 hours in the presence of a catalytic amount of conc. H₂SO₄ (2.20mL, 7.63 mmol). The reaction mixture was allowed to cool to roomtemperature and neutralised with Et₃N (3.0 mL, 76.3 mmol). The reactionmixture was concentrated in vacuo and the residue redissolved in ethylacetate (200 mL), washed with brine (1×50 mL), dried (MgSO₄) and excesssolvent removed under reduced pressure to afford a colourless gum (21.17g, 99%). [α]²⁰ _(D)=−59.4° (c 0.014, CHCl₃). ¹H NMR (CDCl₃): δ2.04-2.08and 2.24-2.35(m, 1H, rotamers of H1, 1:1), 2.64 (bs, 1H, OH), 3.54 and3.74 (s, 3H, rotamers of OMe, 1:1), 3.66-3.69 (m, 2H, H3), 4.47-4.50 (m,2H, H2, H11a), 5.07-5.13 (m, 2H, H6), 7.26-7.35 (m, 5H, H arom). ¹³C NMR(CDCl₃): rotamer ratio 1:1, δ37.8 and 38.5 rotamers of (C1), 51.8 and52.0 rotamers of (OMe), 54.1 and 54.7 rotamers of (C3), 57.4 and 57.7rotamers of (C2), 66.9 and 67.0 rotamers of (C6), 68.6 and 69.3 rotamersof (C11a), 127.0, 127.3, 127.4, 127.7, 127.8, 128.0 and 128.1 rotamersof (C arom). IR (thin film): ν=3435 (OH), 3033, 2953 (OH), 1750 (ester),1680 (C═O), 1586, 1542, 1498, 1422, 1357 (CO₂H), 1170, 1124, 1084, 1052(C—O), 1004, 963, 916, 823, 770, 750, 699, 673 cm⁻¹. MS (FAB) m/z(relative intensity): 280 (M^(+.), 24), 236 (20), 234 (4), 216 (8), 214(4), 213 (2), 206 (2), 204 (7), 203 (4), 202 (10), 201 (2), 181 (5), 144(16), 102 (23), 91 (100). HRMS calcd. for C₁₄H₁₇NO₅=279.1107 found279.1192.

(2S,4R)-N-(Benzoxycarbonyl)-2-hydroxymethyl-4-hydroxyproline (47)

Lithium borohydride (1.57 g, 73 mmol) was added portionwise to asolution of(2S,4R)-N-(benzoxycarbonyl)-2-methyoxycarbonyl-4-hydroxyproline (46)(20.17 g, 73 mmol) in THF (350 mL) at 0° C. The reaction mixture wasallowed to warm to room temperature and stir overnight. The resultingsuspension was cooled to 0° C. and quenched with water (2-3 mL) untileffervescence ceased, at which point 2 M HCl (15 mL) was added todissolve the precipitate. The product was extracted with ethyl acetate(3×150 mL) and the combined organic phases washed with brine (1×100 mL)and then dried (MgSO₄). Concentration in vacuo afforded a white gum(18.25 g, 100%). [α]^(22.3) _(D)=−404° (C=0.043, CHCl₃). ¹H NMR (CDCl₃):δ1.24-1.26 (m, 1H, H1), 1.73-2.08 (m, 1H, H1), 3.40-4.30 (m, 6H, H2, H3,H11, H11a), 5.06 (bs, 1H, OH), 5.09 (s, 2H, H6) 7.25-7.31 (m, 5H, Harom). ¹³C NMR (CDCl₃): δ36.7 (C1), 55.2 (C3), 58.7 (C2), 65.0 (C11),67.0 (C6), 68.7 (C11a), 127.0, 127.5 (C arom), 127.8 (C arom), 128.2 (Carom). IR (thin film): ν=3390 (OH), 3065, 3033, 2953 (OH), 1681 (C═Ocarbamate), 1586, 1538, 1498, 1454, 1192, 1122, 978, 914, 862, 770, 698,673 cm⁻¹. MS (FAB) m/z (relative intensity): 252 (M^(+.), 58), 208 (33),176 (5), 144 (6), 118 (8), 116 (7), 92 (13), 91 (100). HRMS calcd. forC₁₃H₁₇NO₄=251.1158 found 251.1230.

(2S,4R)-N-Benzoxycarbonyl-2-t-butyldimethylsilyloxymethyl-4-hydroxypyrrolidine(48)

t-butyldimethylsilyl chloride (5.78 g, 38.3 mmol) and1,8-diazabicyclo[5,4,0]undec-7-ene (1.44 mL, 9.6 mmol) were added to asolution of alcohol (47) (12.51 g, 49.8 mmol) and triethylamine (7.0 mL,49.8 mmol) in dry DCM (200 mL) which had been allowed to stir for 15minutes at room temperature. The resulting mixture was allowed to stirat room temperature for 18 hours and then diluted with ethyl acetate(300 mL). The organic phase was washed with aqueous saturated ammoniumchloride (2×100 mL) and brine (1×100 mL) dried (MgSO₄) and the solventremoved under reduced pressure to yield a colourless viscous oil (9.84g, 70%). [α]^(22.3) _(D)=−263° (c 0.70, CHCl₃). ¹H NMR (CDCl₃): δ−0.05and −0.06(s, 6H, rotamers of H1′, H2′, 1:1), 0.83 and 0.85 (s, 9H,rotamers of H3′, H5′, H6′, 1:1), 1.95-2.22 (m, 2H, H1,), 2.78 (bs, 1H,OH), 3.44-3.68 (m, 3H, H3, H11), 3.99-4.10 (m, 1H, H2), 4.43-4.46 (m,1H, H11a), 5.11-5.16 (m, 2H, H6) 7.34-7.35 (m, 5H, H arom) ¹³C NMR(CDCl₃): rotamer ratio of 1:1, 5-5.50 (C3′, C5′, C6′), 18.15 (C4′),25.83 (C1′, C2′), 36.55 and 37.27 rotamers of (C1), 55.2 and 55.7rotamers of (C3), 57.3 and 57.8 rotamers of (C2), 62.8 and 63.9 rotamersof (C11), 66.6 and 67.0 rotamers of (C6), 69.7 and 70.3 rotamers of(C11a), 127.8 (C arom), 127.9 (C arom), 128.0 (C arom), 128.4 (C arom),128.5 (C arom), 136.5 and 136.8 rotamers of (C7), 154.9 and 155.2rotamers of (C5). IR (thin film): ν=3415 (OH), 3066, 3034, 2953 (OH),2930, 2884, 2857, 1703 (C═O carbamate), 1587, 1498, 1424, 1360 (C—CH₃),1288 (CH₃Si), 1255 (t-Bu), 1220, 1195 (t-Bu), 1118 (Si—O), 1057, 1003,917, 836, 774, 751, 698, 670 cm⁻¹. MS (EI/CI) m/e (relative intensity):366 (M^(+.), 100), 308 (14), 258 (2), 91 (2).

(2S,4R)-2-t-butyldimethylsilyloxymethyl-4-hydroxypyrrolidine (2)

A slurry of 10% Pd/C (190 mg) in ethyl acetate (20 mL) was added to asolution of TBDMS ether (48) (1.90 g, 5.19 mmol) in ethanol (100 mL).The reaction mixture was hydrogenated (Parr apparatus) for 16 h. Thecatalyst was removed by vacuum filtration through Celite and excesssolvent was evaporated under reduced pressure to give a yellow oil inquantitative yield (1.20 g, 100%). [α]²² _(D)=+35.6° (c 0.042, CHCl₃).¹H NMR (CDCl₃): δ-(0.07-0.08) (m, 6H, H1′, H2′), 0.82 (s, 9H, H3′, H4′,H5′), 1.68-1.73 (m, 2H, H1,), 2.99-3.11 (m, 2H, H11), 3.47-3.50 (m, 3H,H11a, H3), 4.09 (bs, 1H, NH or OH), 4.32 (bs, 1H, NH or OH). ¹³C NMR(CDCl₃): δ−5.4 (C3′, C5′, C6′), 18.1 (C4′), 25.8 (C1′, C2′), 37.4 (C1),54.6 (C11), 58.1 (C2), 64.6 (C3), 72.2 (C11a). IR (thin film): ν=3330(OH), 2928, 2857, 1557, 1421, 1331 (C—CH₃), 1249 (CH₃—Si), 1204 (t-Bu),1191 (t-Bu), 1100 (Si—O), 1073, 993, 713 cm⁻¹. MS (CI) m/e (relativeintensity): 232 (M^(+.), 100), 230 (13), 174 (5), 133 (6), 86 (6).

1,1′-[[(Propane-1,3-diyl)dioxy]bis[2-nitro-5-methoxy-1,4-phenylene)carbonyl]]-bis(2S,4R)-2-t-butyldimethylsilyloxymethyl-4-hydroxypyrrolidine (49)

A catalytic amount of DMF (2 drops) was added to a stirred suspension ofbis-nitroacid (44) (2.00 g, 4.28 mmol) and oxalyl chloride (0.94 mL,10.70 mmol) in dry THF (20 mL), and the reaction mixture was allowed tostir for 4 h. After evaporation of excess THF in vacuo, the resultantyellow residue was dissolved in dry THF (20 mL) and added dropwise overa period of 25 minutes to a vigorously stirred suspension of amine (2)(2.47 g, 10.70 mmol), Et₃N (2.50 mL, 17.9 mmol) and ice/water (0.6 mL)cooled in an ice bath. The mixture was then allowed to warm to roomtemperature for a further 1.5 h. After removal of the THF by evaporationin vacuo, the residue was diluted with water (100 mL) and extracted withethyl acetate (3×100 mL). The combined organic phase was washed withwater (3×25 mL) and brine (3×25 mL), dried (MgSO₄), and the solventremoved in vacuo to afford a yellow oil which was purified by flashchromatography (3% MeOH/CHCl₃) to afford the bis-amide (49) as a yellowsolid (2.05 g, 54%). [α]^(23.8) _(D)=−993° (c 0.033, CHCl₃). ¹H NMR(CDCl₃): δ−0.05 (s, 12H, H1′, H2′), 0.80 (s, 18H, H3′, H5′, H6′),1.96-1.99 (m, 2H, H1), 2.14-2.16(m, 2H, H1), 2.19-2.24 (m, 2H, H13),2.85-2.89 (m, 2H, H2) 3.16-3.19 (m, 4H, H11), 3.63-3.66 (m, 4H, H3),3.81 (s, 6H, OMe), 3.99-4.10 (m, 2H, H3), 4.23 (t, 4H, J=5.3 Hz, Hi2),4.38 (bs, 2H, OH); 5.20-5.25 (m, 2H, H11a), 6.65 (s, 2H, H6), 7.55 (s,2H, H9). ¹³C-NMR (CDCl₃): 5-5.35 (C1′, C2′), 18.2 (C4′), 25.8 (C3′, C5′,C6′), 28.9 (C13), 36.1 (C1), 54.9 (CH₃O), 56.6 (C4), 57.3 (C12), 65.0(C3), 70.0 (C2), 108.0 (C6), 109.4 (C9), 128.2 (Q), 137.2 (Q), 148.1(Q), 148.5 (Q), 154.5 (Q), 166.5 (Q). IR (thin film): ν=3392 (OH), 2950,2856, 1623 (C═O), 1577 (C arom), 1524 (NO₂), 1459, 1432, 1381, 1338(C—CH₃), 1278 (CH₃—Si), 1219 (t-Bu), 1184 (t-Bu), 1075 1053, 1004, 938,914, 837, 778, 724, 668, 649, cm⁻¹. MS (FAB) m/z (relative intensity):894 (M^(+.), 8), 893 (19), 878 (6), 835 (2), 779 (9), 761 (6), 517 (3),459 (5), 258 (7), 100 (3), 86 (4), 75 (29), 73 (100), 59 (17), 58 (6).

1,1′-[[(Propane-1,3-diyl)dioxy]bis[2-amino-5-methoxy-1,4-phenylene)carbonyl]]-bis[(2S,4R)-2-t-butyldimethylsilyloxymethyl-4-hydroxypyrrolidine(50)

A slurry of 10% Pd/C (155 mg) in ethyl acetate (20 mL) was added to asolution of the bis-amide (49) (1.55 g, 1.73 mmol) in ethanol (100 mL).The reaction mixture was hydrogenated (Parr apparatus) for 16 h. Thereaction mixture was filtered through Celite and the solvent was removedunder reduced pressure to give a yellow oil (50) in quantitative yield(1.44 g, 100%). ¹H NMR (CDCl₃): δ0.00 (s, 12H, H1′, H2′), 0.88 (s, 18H,H3′, H5′, H6′), 2.00-2.25 (m, 6H, H1, H13), 3.50-3.72 (m, 12H, H2, H3,H11, H11a), 3.74 (s, 6H, OMe), 4.16-4.20 (m, 4H, H3), 4.30-4.35 (m, 4H,H12), 4.49 (bs, 2H, OH); 6.23 (S, 2H, H9), 6.64 (s, 2H, H6) ¹³C-NMR(CDCl₃): 3-5.5 (C1′, C2′), 18.1 (C4′), 25.8 (C3′, C5′, C6′), 29.6 (C13),35.2 (C1), 56.7 (CH₃O), 62.2 (C4), 64.1 (C3), 70.0 (C2), 102.2 (C9),112.6 (C6), 140.4 (Q), 141.1 (Q), 150.6 (Q), 170.1 (Q); IR (neat):ν=3359 (OH), 2929, 2856, 1621 (C═O), 1591 (C arom), 1469, 1433, 1406,1358, 1346 (C—CH₃), 1261 (CH₃—Si), 1232 (t-Bu), 1175 (t-Bu), 1117, 1056,1006, 866, 835, 776 cm⁻¹. MS (FAB) m/z (relative intensity): 834(M^(+.), 13), 833 (18), 773 (9), 602 (13), 399 (7), 371 (34), 232 (9),206 (22), 192 (14), 176 (13), 166 (44), 150 (8), 100 (10), 73 (100).

1,1′-[[(Propane-1,3-diyl)dioxy]bis[2-amino-N-allyloxycarbonyl-5-methoxy-1,4-phenyl-ene)-carbonyl]]-bis[(2S,4R)-2-t-butyldimethylsilyloxymethyl-4-hydroxy-pyrrolidine(51)

A solution of the bis-amide (50) (2.76 g, 3.31 mmol) and pyridine (1.10mL, 13.60 mmol) in dried DCM (100 mL) was cooled to 0° C. Allylchloroformate (0.80 mL, 7.53 mmol) in DCM (50 mL) was added dropwise andthe resulting mixture allowed to warm to room temperature and stirredfor 16 h. The reaction mixture was diluted with DCM (200 mL) and washedwith 1 M CuSO₄ (3×50 mL), water (1×50 mL) and brine (1×50 mL) beforedrying (MgSO₄). Evaporation of the solvent under reduced pressurefollowed by flash column chromatography (2.5% MeOH/DCM) afforded (51) asa yellow solid (3.24 g, 97%). [α]^(21.1) _(D)=−571° (c 0.007, CHCl₃). ¹HNMR (CDCl₃): δ0.00 (s, 12H, H1′, H2′), 0.89 (s, 18H, H3′, H5′, H6′),2.03-2.36 (m, 6H, H1, H13), 3.51-3.58 (m, 6H, H2, H3), 3.77 (s, 6H,OMe), 4.20-4.26 (m, 8H, H11, H12), 4.28-4.30 (m, 2H, H11a), 4.56-4.60(m, 6H, H8′, OH), 5.25 (dd, J_(1,2)=1.5 Hz, J_(1,3)=15.0 Hz, 4H, H10′),5.90-5.95 (m, 2H, H9′), 6.73 (s, 2H, H6), 7.63 (s, 2H, H9), 8.80 (s, 2H,NH). ¹³C NMR (CDCl₃): 5-5.42 (C1′, C2′), 25.8 (C3′, C5′, C6′), 29.2(C13), 35.4 (C1), 56.3 (CH₃O), 57.1 (C11a), 59.8 (C11), 62.2 (C3), 65.1(C12), 65.7 (C8′), 70.5 (C2), 106.3 (C9), 111.5 (C6), 116.5 (Q), 118.1(C10′), 131.7 (Q), 132.5 (C9′), 144.3 (Q), 150.3 (Q), 153.8 (Q), 169.5(Q). IR (neat): ν=3351 (OH), 2931, 2857, 1762 (Alloc C═O), 1722, 1603(C═O), 1521 (C arom), 1463, 1404, 1264 (CH₃—Si), 1222 (t-Bu), 1106(t-Bu), 1053, 1015, 936, 872, 837, 775, 629, cm⁻¹.

1,1′-[[(Propane-1,3-diyl)dioxy]bis[2-amino-N-allyloxycarbonyl-5-methoxy-1,4-phenylene)-carbonyl]]-bis[(2S)-2-t-butyldimethylsilyloxymethyl-4-oxo-pyrrolidine(52)

A solution of dimethyl sulphoxide (2.10 mL, 28.5 mmol) in dry DCM (20mL) was added dropwise over a 15 minutes period to a stirred, cooled(−45° C.) solution of oxalyl chloride (1.27 mL, 14.60 mmol) in DCM (30mL). After 35 minutes, a solution of alcohol (51) (2.54 g, 2.53 mmol) inDCM (20 mL) was added dropwise over a period of 15 minutes to thereaction mixture at −45° C. After 45 minutes a solution of triethylamine(5.75 mL, 40.3 mmol) in DCM (20 mL) was added over a period of 15minutes and the reaction mixture stirred at −45° C. for 30 minutesbefore warming to room temperature over 45 minutes. The mixture was thenwashed with 1 M CuSO₄ (3×50 mL), water (2×50 mL) and brine (1×50 mL)before drying (MgSO₄) and concentrating in vacuo to give (52) as ayellow solid (2.46 g, 97%). ¹H NMR (CDCl₃): δ0.00 (s, 12H, H1′, H2′),0.86 (s, 18H, H3′, H5′, H6′), 2.50-2.63 (m, 6H, H1, H13), 3.63-3.70 (m,4H, H3), 3.80 (s, 6H, OMe), 3.93-3.97 (m, 6H, H₁₁, H11a), 4.29-4.32 (m,4H, H12), 4.62 (d, 4H, J=5.7 Hz, H8′), 5.27-5.32 (m, 4H, H10′),5.98-6.03 (m, 2H, H9′), 6.74 (s, 2H, H6), 7.74 (s, 2H, H9), 8.80 (s, 2H,NH). ¹³C NMR (CDCl₃): δ−5.76 (C1′, C2′), 18.0 (C4′), 25.7 (C3′, C5′,C6′), 28.8 (C13), 39.6 (C1), 55.0 (C3), 56.4 (CH₃O), 65.3 (C12), 65.8(C8′), 105.9 (C9), 110.7 (C6), 118.2 (C10′), 132.4 (C9′), 150.7 (Q),153.5 (Q), 169.1 (Q), 210.0 (C2). IR (neat): ν=3308 (OH), 2931, 2856,1765 (Alloc C═O), 1730, 1624 (C═O), 1602 (C═O), 1522 (C arom), 1468,1407, 1332, 1259 (CH₃—Si), 1204 (t-Bu), 1105 (t-Bu), 1053, 1010, 937,870, 837, 808, 778, 674, 657 cm^(−1.)

1,1′-[[(Propane-1,3-diyl)dioxy]bis[2-amino-N-allyloxycarbonyl-5-methoxy-1,4-phenylene)-carbonyl]]-bis[(2S)-2-t-butyldimethylsilyloxymethyl-4-methoxycarbonylmethyl-2,3-dihydropyrrole (53)

A solution of diethylmethylphosphonoacetate (0.80 mL, 4.21 mmol) in THF(50 mL) was added to a suspension of NaH (343 mg, 4.21 mmol, 60%dispersion in mineral oil, washed with petroleum ether) in dry THF (50mL) at 0° C. under a nitrogen atmosphere. After stirring at roomtemperature for 1 h, a solution of the dimer ketone (52) (2.04 g, 2.00mmol) in THF (50 mL) was added dropwise at 0° C. The reaction mixturewas allowed to warm to room temperature over 18 h. Excess THF wasremoved under reduced pressure and the residue cooled in an ice bathbefore adding NaHCO₃ (50 mL) followed by EtOAc (50 mL). The layers wereseparated and the aqueous layer washed with EtOAc (2×50 mL). Thecombined organic layers were washed with brine (1×50 mL), dried (MgSO₄)and the solvent removed in vacuo to give a yellow oil. Flash columnchromatography (2.5% MeOH/CH₂Cl₂) afforded the product (53) as a yellowsolid (2.00 g, 88%). ¹H NMR (CDCl₃): δ−0.01 (s, 12H, H1′, H2′), 0.83 (s,18H, H3′, H5′, H6′), 2.35-2.40 (m, 2H, H13), 2.65-2.86 (m, 4H, H1),3.03-3.09 (m, 4H, H14), 3.62 (s, 3H, OMe), 3.75 (s, 6H, H16), 3.95-4.10(m, 4H, H₁₁), 4.24-4.35 (m, 4H, H12), 4.58-4.70 (m, 6H, H8′, H11a),5.25-5.33 (m, 4H, H10′), 5.93-5.97 (m, 2H, H9′), 6.33-6.40 (m, 2H, H3),6.74 (s, 2H, H6), 7.80 (s, 2H, H9), 8.75 (s, 2H, NH). ¹³C NMR (CDCl₃):δ−5.52 (C1′, C2′), 18.0 (C4′), 25.7 (C3′, C5′, C6′), 28.7 (C13), 33.8(C14), 34.6 (Cl), 51.9 (CH₃O), 56.5 (C16), 62.2 (C11), 65.2 (C12), 65.6(C8′), 105.4 (C9), 111.9 (C6), 117.9 (C10′), 128.2 (C3), 132.5 (C9′),143.9 (Q), 150.7 (Q), 153.4 (Q), 165.7 (Q), 170.6 (Q). IR (neat): ν=3402(OH), 2954, 2857, 1735 (ester), 1726 (Alloc C═O), 1642, 1600, 1526 (Carom), 1469, 1435, 1354, 1256 (CH₃—Si), 1221, 1201 (t-Bu), 1112 (t-Bu),1048, 1010, 934, 866, 836, 776 cm⁻¹. MS (FAB) m/z (relative intensity):No parent ion, 496 (10), 482 (9), 455 (11), 441 (13), 232 (12), 206(19), 204 (10), 200 (14), 192 (34), 188 (23), 172 (33), 165 (18), 152(17), 150 (16), 149 (100), 147 (17), 140 (20), 131 (18), 103 (22), 91(47), 89 (27), 87 (36), 80 (33), 75 (42), 73 (77), 61 (39), 57 (53).

1,11-[[(Propane-1,3-diyl)dioxy]bis[2-amino-N-allyloxycarbonyl-5-methoxy-1,4-phenylene)-carbonyl]]-bis[(2S)-2-hydroxymethyl-4-methoxycarbonylmethyl-2,3-dihydropyrrole(54)

Hydrofluoric acid.pyridine complex (3.5 mL) was added to a solution ofdimer ester (53) (740 mg, 0.67 mmol) in THF (10 mL) under a nitrogenatmosphere at 0° C. The reaction was allowed to stir for 30 minutes at0° C. and then to warm to room temperature over 1 h. The reactionmixture was neutralised with NaHCO₃ until evolution of CO₂ ceased. Theproduct was extracted with DCM (3×30 mL), washed with brine (1×20 mL)and then dried (MgSO₄). Removal of solvent under reduced pressure gavethe product as a yellow gum (530 mg, 90%). ¹H NMR (CDCl₃): δ2.39 (m, 2H,H13), 2.95-2.99 (m, 4H, H1), 3.09-3.12 (m, 4H, H14), 3.68 (s, 3H, OMe),3.74-3.78 (m, 4H, H11), 3.81 (s, 6H, H16), 4.28-4.34 (m, 4H, H12), 4.62(d, J=5.5 Hz, 4H, H8′), 4.73-4.75 (m, 2H, H11a), 5.31-5.38 (m, 4H,H10′), 5.96-6.02 (m, 2H, H9′), 6.39-6.50 (m, 2H, H3), 6.80 (s, 2H, H6),7.72 (s, 2H, H9), 8.57 (s, 2H, NH). ¹³C NMR (CDCl₃): δ28.8 (C13), 33.5(C14), 35.5 (C1), 52.1 (CH₃O), 56.6 (C16), 65.3 (C12), 66.0 (C8′), 105.6(C9), 111.8 (C6), 118.1 (CO1′), 128.1 (C3), 132.5 (C9′), 144.4 (Q),151.0 (Q),153.6 (Q), 167.3 (O), 170.7 (Q). IR (neat): ν=3416 (OH), 2953,1731 (ester), 1726 (Alloc C═O), 1606, 1525 (C arom), 1467, 1434, 1358,1224, 1048, 938, 870, 768 cm⁻¹. MS (FAB) m/z (relative intensity): 881(M^(+.), 0.2), 496 (12), 482 (15), 456 (14), 442 (13), 232 (23), 206(35), 192 (63), 190 (21), 188 (17), 180 (19), 178 (25), 152 (39), 150(23), 149 (100), 140 (50), 136 (21), 112 (23), 108 (23), 94 (29), 91(32), 87 (24), 80 (70), 73 (28), 57 (30).

1,1-[[(Propane-1,3-diyl)dioxy]bis[(11aS)-7-methoxy-10-allyloxycarbonyl-(2S)-2-methoxycarbonylmethyl-2,3-dihydropyrrole-1,3,11a-trihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(55)

A solution of dimethyl sulphoxide (0.27 mL, 3.82 mmol) in dried DCM (10mL) was added dropwise over a 15 minutes period to a stirred, cooled(−45° C.) solution of oxalyl chloride (0.17 mL, 1.92 mmol) in DCM (10mL). After 35 minutes, a solution of substrate (54) (600 mg, 0.68 mmol)in DCM (10 mL) was added dropwise over a period of 15 minutes to thereaction mixture at −45° C. After 45 minutes a solution of triethylamine(0.78 mL, 5.42 mmol) in DCM (10 mL) was added over a period of 15minutes and the reaction mixture stirred at −45° C. for 30 minutesbefore being allowed to warm to room temperature over 45 minutes. Themixture was then diluted with water (10 mL) and the layers separated.The organic layer was washed with 1 M HCl (3×50 mL), and brine (1×50 mL)before drying (MgSO₄) and concentrating in vacuo. Flash columnchromatography (1.5% MeOH/CH₂Cl₂) afforded a yellow glass (457 mg, 78%).[α]^(20.3) _(D)=+69° (c 0.484, CHCl₃). ¹H NMR (CDCl₃): δ2.35-2.63 (m,2H, H13), 2.75-3.10 (m, 4H, H1), 3.14-3.19 (m, 4H, H14), 3.71 (s, 3H,OMe), 3.88 (s, 6H, H16), 4.21-4.40 (m, 4H, H12), 4.45-4.50 (m, 2H,H11a), 4.60-4.62 (m, 4H, H8′), 5.26-5.30 (m, 4H, H10′), 5.77 (d, J=8.61Hz, 4H, H11) 5.90-5.96 (m, 2H, H9′), 6.75-6.80 (m, 2H, H3), 6.89 (s, 2H,H9), 7.22 (s, 2H, H6). ¹³C NMR (CDCl₃): δ28.8 (C13), 33.5 (C14), 35.5(C1), 52.1 (CH₃O) 56.6 (C16), 65.3 (C12), 66.0 (C8′), 105.6 (C9), 111.8(C6), 118.1 (C10′), 128.1 (C3), 132.5 (C9′), 144.4 (Q), 151.0 (Q),153.6(Q), 167.3 (Q), 170.7 (Q). IR (neat): ν=3583, 3412 (OH), 1730 (ester),1713 (Alloc C═O), 1644, 1421, 1362, 1273, 1223, 1092, 902, 757, 737,702, 667 cm⁻¹. MS (FAB) m/z (relative intensity): 907 (M^(+.), 1), 456(6), 245 (7), 232 (16), 218 (13), 206 (23), 205 (10), 204 (14), 192(42), 190 (17), 178 (22), 177 (10), 176 (16), 166 (17), 165 (10), 164(16), 152 (23), 151 (12), 150 (18), 149 (100), 140 (16), 93 (18), 91(22), 89 (13), 87 (26), 80 (58), 75 (19), 73 (28), 57 (25).

1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11aS)-7-methoxy-(2S)-2-methoxycarbonylmethyl-2,3-dihydropyrrole-1,3,11a-trihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(56)

A catalytic amount of tetrakistriphenylphosphinepalladium(0) (16 mg,0.014 mmol) was added to a solution of carbinolamine (55) (219 mg, 0.25mmol), triphenylphosphine (7 mg, 0.025 mmol) and pyrrolidine (0.05 mL,0.80 mmol) in dry DCM (30 mL) at 0° C. The reaction mixture was stirredfor 2 hours before being allowed to warm to room temperature over 1 h.The solvent was removed in vacuo and the residue was subjected to flashcolumn chromatography (2% MeOH/CH₂Cl₂₁ R_(f)=0.25) to yield a yellowglass (109 mg, 66%). [α]^(19.5) _(D)=+500° (c 0.043, CHCl₃). ¹H NMR(CDCl₃): δ2.17-2.42 (m, 2H, H13), 3.15-3.32 (m, 8H, H1, H14), 3.73 (s,3H, OMe), 3.91 (s, 6H, H16), 4.26-4.30 (m, 6H, H12, H11a), 6.84 (s, 2H,H9), 6.92-7.06 (m, 2H, H3), 7.47 (s, 2H, H6), 7.83 (d, J=4.0 Hz, 4H,H11). ¹³C NMR (CDCl₃): δ28.7 (C13), 33.6 (C14), 37.4 (C1), 52.2 (CH₃O),53.8 (C11), 56.2 (C16), 65.4 (C12), 110.9 (C9), 111.8 (C6), 126.5 (C3),140.2 (Q), 148.0 (Q), 151.0 (Q), 161.4 (Q), 162.6 (C11a), 170.7 (Q). IR(neat): ν=3583, 3394, 2997, 2950, 1736 (ester), 1717 (Alloc C═O), 1628,1596, 1511, 1483, 1451, 1431, 1382, 1273, 1245, 1197, 1152, 1068, 995,963, 914, 842, 753 cm⁻¹. FABMS m/z (relative intensity): 673 (M^(+.),2), 279 (6), 277 (4), 201 (7), 185 (55), 181 (7), 110 (5), 93 (100), 91(24), 75 (28), 73 (20), 61 (12), 57 (33).

Example 1(v)

Synthesis of(11aS)-1,11a-dihydro-7,8-dimethoxy-2-ethenyl-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(See FIGS. 6 a/b)

DRH360 N-(4,5-dimethoxy-2-nitrobenzoyl)hydroxyproline Methyl Ester (169)

Oxalyl chloride (15.38 g, 121.11 mmol) was added in one portion to astirred suspension of 2-nitro-4,5-dimethoxybenzoic acid (34) (25.01 g,110.10 mmol) in anhydrous DCM (100 mL) at room temperature. A catalyticamount of DMF (2 drops) was added (CARE!—increased gas evolution) andthe reaction mixture was allowed to stir for 16 hours under an inertatmosphere. The acid chloride solution was added dropwise to avigorously stirred solution of the pyrrolo C-ring (168) (34.90 g, 110.10mmol, JOC 5, 13, 1994, 3621) and TEA (45.95 mL, 33.36 g, 330.29 mmol) inanhydrous DCM (100 mL) at −20° C. The reaction mixture was allowed tostir for 16 hours at room temperature. The reaction mixture was washedwith saturated NaHCO₃ (2×200 mL), saturated NH₄Cl (2×200 mL), water(2×200 mL), brine (2×200 mL) and dried over anhydrous MgSO₄. Filtrationand evaporation of the solvent in vacuo afforded the crude product(169), which was purified by flash column chromatography using EtOAc aseluent. Pure fractions were combined and evaporation of excess eluent invacuo afforded the product as a foam (33.26 g, 93.9 mmol, 85%). ¹H NMR(270 MHz, CDCl₃) d 7.69 (s, 1H), 6.87 (s, 1H), 5.31 (s, 2H), 4.97-4.82(m, 1H), 4.44 (br s, 1H), 3.99 (s, 3H), 3.98 (s, 3H), 3.81 (s, 3H),3.54-3.48 (m, 1H), 3.18 (d, 1H, J=2.02 Hz), 2.87 (br s, 1H), 2.45-2.16(m, 2H); ¹³C NMR (67.8 MHz, CDCl₃) d 172.6, 172.5, 167.5, 166.8, 154.4,154.0, 149.3, 137.5, 137.4, 127.0, 126.2, 109.5, 107.2, 107.1, 69.9,69.1, 59.2, 57.4, 56.9, 56.8, 56.6, 56.4, 54.6, 53.5, 52.5, 52.4, 39.4,38.0.

(11aS)-6,7-dimethoxy-2(R)-hydroxy-2,3,5,10,11,11a-hexahydro-5,11-dioxo-1H-pyrrolo[2,1-c][1,4-]benzodiazepine(170)

10% Pd/C catalyst (3.3 g) was added to a solution of 169 (33.0 g, 93.1mmol) in absolute EtOH (250 mL). The reaction mixture was hydrogenatedunder pressure using a Parr hydrogenator at 55 psi H₂ for 18 h. Thereaction mixture was filtered through celite, and the celite washed withhot MeOH, taking care not to allow the filter cake to dry out. Removalof excess solvent afforded the crude product (20.14 g). The crudeproduct was allowed to stir in 1 N HCl (200 mL) and CHCl₃ (200 mL) for30 minutes. The organic layer was washed with 1 N HCl (100 mL) and theaqueous layers were combined and neutralised with saturated aqueousNaHCO₃. On leaving the aqueous extract overnight, a fine whiteprecipitate formed (170) which was collected by filtration and dried(7.81 g, 26.72 mmol, 29%). ¹H NMR (270 MHz, CDCl₃) d 10.06 (s, 1H, NH),7.61 (s, 1H, ArH), 7.36 (s, 1H, ArH), 4.49-4.41 (m, 1H, 2), 4.22-4.17(m, 1H, 11a), 3.88 (s, 6H), 3.82-3.55 (m, 2H, 3), 3.20 (br s, 1H, OH),2.87-2.77 (m, 1H, 1), 2.10-2.05 (m, 1H, 1); ¹³C NMR (CDCl₃) d 170.2,165.9, 152.0, 145.7, 130.7, 118.2, 111.9, 104.2, 68.1, 56.0, 55.6, 54.2,34.6, 18.8.

(11aS)-6,7-dimethoxy-2(R)-[(tert-butyldimethylsilyl)oxy]-2,3,5,10,11,11a-hexahydro-5,11-dioxo-1H-pyrrolo[2,1-c][1,4-]benzodiazepine(171)

Solid TBDMS Chloride (8.22 g, 54.44 mmol) was added in one portion to asolution of 170 (7.23 g, 24.74 mmol) and imidazole (8.42 g, 123.72 mmol)in anhydrous DMF (75 mL) and allowed to stir at room temperature for 16h. The reaction mixture was poured into water (500 mL) and filtered toafford the crude product (171), which was purified by recrystallisationfrom EtOH (800 mL) as fine white needles (6.995 g, 17.21 mmol, 70%). ¹HNMR (270 MHz, CDCl₃) δ10.06 (s, 1H, NH), 7.37 (s, 1H, ArH), 6.68 (s, 1H,ArH), 4.19-4.14 (m, 1H, 2), 4.06-4.01 (m, 1H, 11a), 3.90 (s, 3H, OMe),3.88 (s, 3H, OMe), 3.69-3.63 (m, 2H, 3), 2.85-2.80 (m, 1H, 1), 2.05-2.01(m, 1H, 1); ¹³C NMR (67.8 MHz, CDCl₃) δ170.4, 170.2, 165.9, 152.1,145.8, 145.6, 131.1, 130.7, 118.1, 111.9, 104.3, 104.1, 69.2, 69.1,56.0, 55.9, 55.7, 54.3, 54.0, 35.0, 25.8, 25.7, 25.6, 17.9, −3.0, −3.5,−4.9, −5.0.

(11aS)-6,7-dimethoxy-2(R)-[(tert-butyldimethylsilyl)oxy]-2,3,5,10,11,11a-hexahydro-10-[2-(trimethylsilyl)ethoxymethyl]-5,11-dioxo-1H-pyrrolo[2,1-c][1,4-]benzodiazepine(172)

A solution of 171 (6.50 g, 15.99 mmol) in anhydrous DMF (27.5 mL) wasadded dropwise to a stirred suspension of NaH (0.422 g, 0.704 g of a 60%dispersion in mineral oil, 18.34 mmol) at 0° C. and the reaction mixturewas allowed to stir for 30 minutes. A solution of SEM chloride (3.11 mL,2.93 g, 17.59 mmol) in anhydrous DMF (5 mL) was added dropwise to thestirred reaction mixture at 0° C. and allowed to stir at roomtemperature for 16 h. The reaction mixture was poured into water (200mL) to afford a white precipitate, which was extracted with diethylether (4×300 mL). The organic layer was washed with water (2×50 mL),brine (2×50 mL) and dried over anhydrous MgSO₄. Filtration andevaporation of the solvent in vacuo afforded the crude product, whichwas purified by flash column chromatography using an 80:20 mixture ofpetroleum ether:EtOAc as eluent. Pure fractions were combined andevaporated in vacuo to afford the product (172) as a yellow oil (7.01 g,13.1 mmol, 82%). ¹H NMR (270 MHz, CDCl₃) δ7.35 (s, 1H, ArH), 7.24 (s,1H, ArH), 5.52 (d, 2H, J=9.89 Hz, SEM amino acetal CH₂), 4.65 (d, 2H,J=9.90 Hz, SEM amino acetal CH₂), 4.61-4.56 (m, 1H, 2), 4.23 (dd, 1H,J=4.40 Hz, 8.24 Hz, 11a), 3.94 (s, 3H, OMe), 3.92 (s, 3H, OMe), 3.68 (m,4H, SEM 1′ CH₂+3), 2.86 (m, 1H, 1), 2.02 (m, 1H, 1), 0.98 (t, 2H, J=8.25Hz, SEM 2′ CH₂), 0.88 (s, 9H, TBDMS t-Bu CH₃), 0.10 (s, 6H, 2×TBDMSSiCH₃), 0.03 (s, 9H, 3× SEM SiCH₃); ¹³C NMR (67.8 MHz, CDCl₃) δ170.0,165.6, 151.8, 147.1, 133.9, 121.5, 111.2, 105.5, 78.1, 69.6, 67.0, 56.5,56.2, 56.1, 53.6, 35.5, 25.7, 18.4, −1.3, −4.8.

(11aS)-6,7-dimethoxy-2(R)-hydroxy-2,3,5,10,11,11a-hexahydro-10-[2-(trimethylsilyl)ethoxymethyl]-5,11-dioxo-1H-pyrrolo[2,1-c][1,4-]benzodiazepine(173)

A solution of 1 N TBAF in THF (19.58 mL, 19.58 mmol) was added to astirred solution of 172 (7.0 g, 13.05 mmol) in THF (50 mL). The reactionmixture was allowed to stir at room temperature for 2 hours and dilutedwith DCM (200 mL), washed with water (2×200 mL), brine (2×200 mL) anddried over anhydrous MgSO₄. Filtration and removal of excess solventafforded the crude product, which was purified by flash columnchromatography using 50:50 petroleum ether:EtOAc as eluent. Evaporationin vacuo of the pure fractions afforded the product (173) (5.9 g). ¹HNMR (270 MHz, CDCl₃) δ7.30 (s, 1H, ArH), 7.24 (s, 1H, ArH), 5.52 (d, 1H,J=9.9 Hz, SEM amino acetal CH₂), 4.68-4.64 (m, 2H, SEM amino acetalCH₂+2), 4.30 (dd, 1H, J=5.86, 8.24 Hz), 3.91 (s, 3H, OMe), 3.90 (s, 3H,OMe), 3.87-3.51 (m, 4H, SEM 1′ CH₂+3), 2.95 (dt, 1H, J=5.31, 13.56 Hz,1), 2.17-2.08 (m, 1H, 1), 1.02-0.93 (m, 2H, SEM 2′ CH₂), 0.03 (s, 9H, 3×SiCH₃); ¹³C NMR (67.8 MHz, CDCl₃) δ169.7, 165.9, 151.9, 147.1, 134.0,121.1, 111.2, 105.5, 78.2, 69.1, 67.1, 56.5, 56.1, 53.9, 34.9, 18.4,−1.3.

(11aS)-6,7-dimethoxy-2,3,5,10,11,11a-hexahydro-10-[2-(trimethylsilyl)ethoxymethyl]-2,5,11-trioxo-1H-pyrrolo[2,1-c][1,4-]benzodiazepine(174)

Anhydrous DMSO (3.28 g, 41.94 mmol) in dry DCM (20 mL) was addeddropwise over 5 minutes to a stirred solution of oxalyl chloride (10.48mL of a 2 N solution in DCM, 20.97 mmol) under a nitrogen atmosphere at−50° C. After stirring for 5 minutess, a solution 173 (5.90 g, 13.98mmol), in dry DCM (45 mL) was added dropwise over 45 minutesutes to thereaction mixture, which was then stirred for a further 45 minutesutes at−50° C. TEA (9.89 g; 97.87 mmol) was added dropwise to the mixture over15 minutes followed by stirring for a further 15 minutes. The reactionmixture was left to warm to room temperature, diluted with H₂O (150 mL)and DCM (100 mL). The organic phase was washed with 1 N HCl (2×100 mL),water (2×100 mL), brine (2×100 mL) and dried over MgSO₄. Filtration andevaporation afforded the crude product (174), which was purified byflash column chromatography using 50:50 petroleum ether (40-60°):EtOAcas eluent. Evaporation of the pure fractions in vacuo afforded theproduct (4.33 g, 10.3 mmol, 74%). ¹H NMR (270 MHz, CDCl₃) δ7.30 (s, 1H,ArH), 7.24 (s, 1H, ArH), 5.60 (d, 1H, J=9.89 Hz, SEM amino acetal CH₂),4.69 (d, 1H, J=9.89 Hz, SEM amino acetal CH₂), 4.62 (dd, 1H, J=9.89,3.12 Hz, 11a), 4.26-4.19 (m, 1H, 3), 3.95 (s, 3H, Ome), 3.94 (s, 3H,OMe), 3.81-3.49 (m, 4H, SEM 1′ CH₂+1+3), 2.82-2.71 (m, 1H, 1), 0.95 (t,2H, J=2.01 Hz, SEM 2′ CH₂), −0.04 (s, 9H, SEM CH₃); ¹³C NNR (67.8 MHz,CDCl₃) δ206.8, 168.8, 165.9, 152.4, 147.5, 134.0, 120.4, 111.1, 105.6,78.2, 67.2, 56.2, 54.8, 52.3, 37.3, 18.3, −1.3.

(11aS)-5,10,11,11a-tetrahydro-7,8-dimethoxy-10-[2-trimethylsilyl)ethoxymethyl]-2-[[(trifluoromethyl)sulphonyl]oxy]-5,11-dioxo-1H-pyrrolo[2,1-c][1,4]benzodiazepine(175)

Anhydrous pyridine (0.46 mL, 0.452 g, 5.73 mmol) was added in oneportion to a vigorously stirred solution of 174 (2.0 g, 4.77 mmol) inanhydrous DCM (100 mL) and the mixture left to stir for 10 minutes atroom temperature. Anhydrous triflic anhydride (1.25 mL, 1.48 g, 5.25mmol) was added quickly, in one portion, and the reaction mixture wasallowed to stir at room temperature for 4.5 h. The darkened, homogenousreaction mixture was poured into cold saturated NaHCO₃ (200 mL) and themixture was extracted with DCM (3×50 mL). The organic layers werecombined, washed with water (2×200 mL), brine (2×200 mL) and dried overanhydrous MgSO₄. Filtration and evaporation afforded the crude product,which was purified by flash column chromatography using 80:20 petroleumether:EtOAc as eluent. Evaporation of the pure fractions in vacuoafforded the product (175) as a yellow oil (1.79 g, 3.25 mmol, 68%). ¹HNMR (270 MHz, CDCl₃) δ7.29 (s, 1H, ArH), 7.23 (s, 1H, ArH), 7.15 (t, 1H,J=2.01 Hz, H3), 5.53 (d, 1H, J=10.07 Hz, SEM amino acetal CH₂), 4.68 (d,1H, J=9.89 Hz, SEM amino acetal CH₂).

(11aS)-7,8-Dimethoxy-2-ethenyl-5,10,11,11a-tetrahydro-10-(2-(trimethylsilyl)ethoxymethyl)-5,11-dioxo-1H-pyrrolo[2,1-c][1,4]benzodiazepine(176)

A catalytic amount of tetrakistriphenylphosphine palladium [0] (4 mol %,0.142 g, 0.123 mmol) was added to a stirred mixture of 175 (1.69 g, 3.06mmol), LiCl (0.39 g, 9.19 mmol), and tributylvinyltin (1.16 mL, 1.26 g,3.98 mmol) in anhydrous THF (100 mL) and heated at reflux for 2.5 h. Thecooled reaction mixture was diluted with DCM (100 mL) and the mixturewashed with 10% aqueous ammonium hydroxide (200 mL). The organic layerwas washed with brine (2×200 mL) and dried over anhydrous MgSO₄.Filtration and evaporation of the solvent in vacuo afforded the crudeproduct, which was further purified by flash column chromatography usinga 80:20 mixture of petroleum ether:EtOAc as eluent. Pure fractions werecombined and evaporation of the solvent in vacuo afforded the product(176) as a colourless oil (0.992 g, 2.312 mmol, 75.5%). %). ¹H NMR (270MHz, CDCl₃) δ7.32 (s, 1H, ArH), 7.22 (s, 1H, ArH), 6.94 (s, 1H, H3),6.51 (dd, 1H, J=10.62, 17.22 Hz, alkene CH), 5.51 (d, 1H, J=10.07 Hz,SEM amino acetal CH₂), 5.20 (d, 1H, J=8.24 Hz, alkene CH₂), 5.15 (s, 1H,alkene CH₂), 4.66 (d, 1H, J=9.89 Hz, SEM amino acetal CH₂), 4.54 (dd,1H, J=3.30, 10.62 Hz, H11a), 3.90 (s, 3H, OMe), 3.89 (s, 3H, OMe),3.82-3.60 (m, 3H, SEM 1′ CH₂+H1), 2.91-2.82 (m, 1H, H1), 0.96 (t, 2H,J=8.42 Hz, SEM 2′ CH₂), −0.04 (s, 9H, SEM CH₃); ¹³C NMR (67.8 MHz,CDCl₃) δ169.3, 161.8, 152.1, 147.3, 133.8, 129.8, 126.0, 125.1, 121.2,115.1, 111.4, 105.9, 78.5, 67.1, 57.6, 56.2, 56.2, 29.6, 18.4, −1.34.

(11aS)-1,11a-Dihydro-7,8-dimethoxy-2-ethenyl-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(177)

Solid sodium tetraborohydride (NaBH₄, 81 mg, 2.175 mmol) was added inone portion to a rapidly stirred solution of 176 (101 mg, 0.233 mmol) ina mixture of anhydrous EtOH (2 mL) and anhydrous THF (4 mL) at roomtemperature and allowed to stir for 4 h. The reaction mixture wasdiluted with water (5 mL) and extracted with CHCl₃ (3×5 mL). The organiclayers were washed with brine (10 mL) and dried over anhydrous MgSO₄.Filtration and evaporation afforded the crude product, which was stirredfor 30 minutes with silica gel (0.25 g) in MeOH (5 mL). Excess methanolwas removed by rotary evaporation, causing the crude product to beabsorbed onto the silica gel. The plug of silica gel was added to thetop of a silica gel column and the product was purified by flash columnchromatography eluting with a 60:40 mixture of petroleum ether:EtOAc.Pure fractions were combined and evaporation of the solvent in vacuoafforded the product (177) as a yellow solid (33 mg, 0.116 mmol, 50%).¹H NMR (270 MHz, CDCl₃) δ7.86 (d, 1H, J=3.84 Hz, imine CH), 7.50 (s, 1H,ArH), 7.05 (br s, 1H, H3), 6.82 (s, 1H, ArH), 6.58 (dd, 1H, J=10.62,17.22 Hz, alkene CH), 5.20-5.05 (m, 2H, alkene CH₂), 4.39-4.31 (m, 1H,H11a), 3.96 (s, 3H, OMe), 3.94 (s, 3H, OMe), 3.39-3.12 (m, 2H, H1); ¹³CNMR (67.8 MHz, CDCl₃) δ162.7, 161.5, 151.9, 147.8, 140.4, 129.9, 126.9,123.9, 118.9, 114.4, 111.6, 109.8, 77.3, 56.2, 53.9, 33.7.

Example 1(h)

Synthesis of(11aS)-1,11a-Dihydro-7.8-dimethoxy-2-(4-methoxyphenyl)-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(See FIGS. 6 a/b)

(11aS)-7,8-Dimethoxy-2-(4-methoxyphenyl)-5,10,11,11a-tetrahydro-10-(2-(trimethylsilyl)ethoxymethyl)-5,11-dioxo-1H-pyrrolo[2,1-c][1,4]benzodiazepine(178)

A solution of para-methoxyphenylboronic acid (301 mg, 1.98 mmol) in DME(10 mL) was added to a stirred solution of vinyl triflate (175—seeexample 1(g)) (715 mg, 1.29 mmol) in DME (10 mL) under a nitrogenatmosphere. An aqueous solution of Na₂CO₃ (2 N, 9.9 mL) was addedfollowed by LiCl (178 mg, 4.185 mmol) andtetrakis(triphenylphosphine)palladium(0) (5 mol %, 81 mg) and themixture was stirred for 1 hour at room temperature followed by heatingat reflux for 1 h. After concentration in vacuo, the residue wasresuspended in a mixture of DCM (50 mL), aqueous 2 N Na₂CO₃ (50 mL) andconc. NH₄OH solution (3 mL). The aqueous layer was extracted with DCM(3×20 mL) and the combined organic extracts were dried over anhydrousMgSO₄. Filtration and evaporation afforded a residue which was purifiedby flash column chromatography on silica gel eluting with 60:40petroleum ether:EtOAc. Pure fractions were combined and evaporation ofthe solvent in vacuo afforded the product (178) as a yellow solid (559mg, 1.095 mmol, 85%). ¹H NMR (270 MHz, CDCl₃) δ7.40-7.35 (m, 3H,ArH+Suzuki ArH), 7.33 (t, 1H, J=2.01 Hz, H3), 7.27 (s, 1H, ArH), 6.88(d, 2H, J=6.93 Hz, Suzuki ArH), 5.56 (d, 1H, J=9.89 Hz, SEM amino acetalCH₂), 4.71 (d, 1H, J=9.89 Hz, SEM amino acetal CH₂), 4.63 (dd, 1H,J=3.30, 10.62 Hz, H11a), 3.95 (s, 3H, Ode), 3.94 (s, 3H, OMe), 3.87-3.65(m, 6H, SEM 1′ CH₂+H1+Suzuki ArOMe), 3.14 (ddd, 1H, J=2.38, 10.62, 16.12Hz, H1), 0.96 (t, 2H, J=8.42 Hz, SEM 2′ CH₂), −0.04 (s, 9H, SEM CH₃);¹³C NMR (67.8 MHz, CDCl₃) d 168.4, 161.6, 160.2, 153.0, 147.3, 133.7,126.5, 126.1, 125.4, 121.3, 120.2, 114.1, 111.3, 105.8, 78.4, 67.1,57.5, 56.2, 56.2, 55.3, 31.5, 18.4, −1.34.

(11aS)-1,11a-dihydro-7,8-Dimethoxy-2-(4-methoxyphenyl)-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(179)

Solid sodium tetraborohydride (NaBH₄, 70 mg, 1.88 mmol) was added in oneportion to a rapidly stirring solution of 178 (100 mg, 0.2 mmol) in amixture of anhydrous EtOH (2 mL) and anhydrous THF (4 mL) and left tostir at room temperature for 9 h. The reaction mixture was diluted withwater (10 mL) and stirred for 30 minutes with silica gel (2.0 g). Themixture was extracted with EtOAc (3×10 mL). The organic layers werewashed with brine (10 mL) and dried over anhydrous MgSO₄. Filtration andevaporation afforded the crude product (179), which was purified byflash column chromatography eluting with a 50:50 mixture of petroleumether:EtOAc. Pure fractions were combined and evaporation of the solventin vacuo afforded the product as a yellow glass (28 mg, 0.08 mmol, 38%).¹H NMR (270 MHz, CDCl₃) δ7.89 (d, 1H, J=4.03 Hz, Imine CH), 7.53 (s, 1H,ArH), 7.39 (t, 1H, J=1.83 Hz, H3), 7.33 (d, 2H, J=8.97 Hz, methoxyphenylArH), 6.91 (d, 2H, J=8.98 Hz, methoxyphenyl ArH), 6.83 (s, 1H, ArH),4.44-4.36 (m, 1H, H11a), 3.97 (s, 3H, OMe), 3.94 (s, 3H, OMe), 3.91-3.79(m, 4H, H1+Suzuki ArOMe), 3.64-3.34 (m, 1H, H1); ¹³C NMR (67.8 MHz,CDCl₃) δ162.7, 161.3, 159.2, 151.8, 147.8, 140.4, 126.3, 126.2, 126.0,125.9, 123.2, 121.9 114.3, 114.1, 111.6, 109.8, 56.2, 56.1, 55.6, 35.6.

Example 1(i)

Synthesis of(11aS)-1,11a-Dihydro-7,8-dimethoxy-2-phenyl-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(See FIGS. 6 a/b)

(11aS)-7,8-Dimethoxy-2-phenyl-5,10,11,11a-tetrahydro-10-(2-(trimethylsilyl)ethoxymethyl)-5,11-dioxo-1H-pyrrolo[2,1-c][1,4]benzodiazepine(180)

Phenylboronic acid (334 mg, 2.74 mmol, 2.54 equiv.), Na₂CO₃ (343.4 mg,3.24, 3 equiv) and tetrakis (triphenylphosphine) palladium(0) (49.9 mg,2% mmol) was added to a solution of the triflate (175—see example l(g))(600 mg. 1.08 mmol) in ethanol (21.6 mL) water (21.6 mL) and thereaction mixture was allowed to stir at room temperature for 2 hrs. Thereaction mixture was diluted with ethyl acetate (200 mL, washed withwater (2×200 mL), brine (200 mL). and dried over magnesium sulphate.Filtration and evaporation of excess solvent afforded the crude product,which was subjected to flash column chromatography on silica gel (70%40-60° petroleum ether; 30% ethyl acetate) to afford, after removal ofexcess eluent, the compound 180 (405 mg, 0.84 mmol, 78% yield). ¹H NMR(270 MHz, CDCl₃) δ7.5-7.1 (m, 8H), 5.53 (d, 1H, J=10.08 Hz), 4.67 (d,1H, J=10.08 Hz) 4.65-4.59 (m, 1H) 4.0-3.60 (m 9H), 3.12 (dd, 1H,J=10.63, 16.12 Hz), 0.99-0.93 (m, 2H) 0.00 (s, 9H); ¹³C NMR (67.8 MHz,CDCl₃) δ168.3, 161.9, 152.1, 147.3, 133.9, 132.7, 128.7, 127.6, 125.7,121.8, 121.1, 111.3, 105.8, 78.4, 67.2, 57.6, 56.2, 31.3, 18.4, −1.3

(11aS)-1,11a-Dihydro-7,8-dimethoxy-2-phenyl-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(181)

Solid sodium tetraborohydride (287 mg, 7.6 mmol, 10 equiv.) was added inone portion to a rapidly stirred solution of 180 (365 mg, 0.76 mmol) ina mixture of anhydrous EtOH (8 mL) and anhydrous THF (8 mL) at 0° C. Thereaction mixture was allowed to stir at room temperature for 1 hour atroom temperature at which time TLC (5% methanol; 95% chloroform)revealed the complete consumption of starting material. The reactionmixture was diluted with ethyl acetate (100 mL), washed with water(2×100 mL), brine (100 mL) and dried over magnesium sulphate. Filtrationand evaporation of excess solvent afforded the crude product as a brownviscous oil. Flash chromatography (silica gel, 70% 40-60° petroleumether, 30% ethyl acetate yield the final product (181) (271 mg, 0.77mmol, 74%). ¹H NMR (270 MHz, CDCl₃) δ7.89 (d, 1H, J=4.03 Hz), 7.53 (s,1H), 7.51 (s, 1H), 7.40-7.20 (m, 5H), 6.83 (s, 1H), 4.50-4.35 (m, 1H),3.96 (s, 3H), 3.94 (s, 3H), 3.66-3.36 (m, 2H). ¹³C NMR (67.8 MHz, CDCl₃)δ162.6, 161.5, 151.9, 147.8, 140.4, 133.3, 128.8, 127.6, 127.1, 124.9,123.6, 119.0, 111.6, 109.8, 56.2, 53.9, 35.4. HRMS (FAB) calcd forC₂₀H₁₉N₂O₃ (M⁺.+1) 335.1398, found 335.1396.

Example 2(a)

Synthesis of the C7-Iodo-C2-methlene PBD Monomer BSD-SJG (64, UP-2023)(See FIG. 7)

(S)-N-(Allyloxycarbonyl)-2-(tert-butyldimethylsilyloxymethyl)-4-methylidenepyrrolidine(57)

Potassium tert-butoxide (41.0 mL of a 0.5 M solution in THF, 20.5 mmol)was added dropwise to a suspension of methyltriphenylphosphonium bromide(7.29 g, 20.4 mmol) in THF (20 mL) at 0° C. (ice/acetone) undernitrogen. After stirring for 2 hours at 0° C., a solution of the ketone16 (example 1(b)) (3.20 g, 10.2 mmol) in THF (10 mL) was added dropwiseand the mixture allowed to warm to room temperature. After stirring fora further 30 minutes the reaction mixture was diluted with EtOAc (150mL) and water (150 mL) and the organic layer separated, washed withbrine, dried (MgSO₄), filtered and evaporated in vacuo to give a yellowoil in which crystals (TPO) formed upon standing in the freezer.Purification by flash chromatography (5% EtOAc/Petroleum Ether) isolatedthe pure olefin 57 as a colourless oil (2.76 g, 87%): [α]²¹ _(D)=−22.2°(c=0.25, CHCl₃); ¹H NMR (270 MHz, CDCl₃) (Rotamers) δ6.02-5.87 (m, 1H,NCO₂CH₂CH═CH₂), 5.31 (ddd, 1H, J=1.65, 3.11, 17.20 Hz, NCO₂CH₂CH═CH₂),5.21 (dd, 1H, J=1.46, 10.40 Hz, NCO₂CH₂CH═CH₂), 4.99-4.61 (m, 2H,NCH₂C═CH₂), 4.60 (d, 2H, J=4.94 Hz, NCO₂CH₂CH═CH₂), 4.19-3.98 (m, 2H,NCHCH₂OTBDMS), 3.93-3 3.87 (m, 1H, NCHCH₂OTBDMS), 3.66-3.42 (m, 2H,NCH₂C═CH₂), 2.80-2.56 (m, 2H, NCH₂C═CH₂CH₂), 0.87 (s, 9H, SiC(CH₃)₃),0.03-0.02 (m, 6H, Si(CH₃)₂); ¹³C NMR (67.8 MHz, CDCl₃) (Rotamers) δ154.4(NC═O), 145.1 and 144.1 (NCH₂C═CH₂), 133.1 (NCO₂CH₂CH═CH₂), 117.5 and117.2 (NCO₂CH₂CH═CH₂), 107.5 and 106.9 (NCH₂C═CH₂), 65.8 and 65.6(NCO₂CH₂CH═CH₂), 63.7 and 63.1 (NCHCH₂OTBDMS), 58.7 and 58.3(NCHCH₂OTBDMS), 51.1 (NCH₂C═CH₂), 34.9 and 34.2 (NCH₂C═CH₂CH₂), 25.8(SiC(CH₃)₃), 18.2 (SiC(CH₃)₃), −5.5 (Si(CH₃)₂); MS (CI), m/z (relativeintensity) 312 (M^(+.) +1, 82), 296 (9), 279 (5), 255 (20), 254 (M—OC₃H₅or M—^(t)Bu, 100), 168 (8), 122 (14); IR (Neat) 3083 (C═CH₂), 2954,2847, 1709 (NC═O), 1533, 1467, 1404 (SiCH₃), 1360, 1310, 1252 (SiCH₃),1207, 1174, 1103, 1076, 1006, 836, 776, 680 cm⁻¹.

(2S)-2-(tert-butyldimethylsilyloxymethyl)-4-methylidenepyrrolidine (58)

A catalytic amount of PdCl₂(PPh₃)₂ (92 mg, 0.131 mmol) was added to asolution of the allyl carbamate 57 (1.0 g, 3.22 mmol) and H₂O (0.34 mL,18.9 mmol) in CH₂Cl₂ (30 mL). After 5 minutes stirring at roomtemperature, Bu₃SnH (0.96 mL, 1.04 g, 3.57 mmol) was added rapidly inone portion. A slightly exothermic reaction with vigorous gas evolutionimmediately ensued. The mixture was stirred for 16 hours at roomtemperature under nitrogen at which point TLC (50% EtOAc/PetroleumEther) revealed the formation of amine. After diluting with CH₂Cl₂ (30mL), the organic solution was dried (MgSO₄), filtered and evaporated invacuo to give an orange oil which was purified by flash chromatography(50-100% EtOAc/Petroleum Ether) to afford the amine 58 as a slightlyorange oil (0.56 g, 77%): [α]²¹ _(D)=−3.9° (c=5.0, CHCl₃); ¹H NMR (270MHz, CDCl₃) δ4.93 (t, 1H, J=2.02 Hz, NCH₂C═CH₂), 4.90 (t, 1H, J=2.02 Hz,NCH₂C═CH₂), 3.68-3.46 (m, 4H, NCHCH₂OTBDMS and NCH₂C═CH₂), 3.30-3.21 (m,1H, NCHCH₂OTBDMS), 2.53-2.41 (m, 2H, NCH₂C═CH₂CH₂ and NH), 2.26-2.17 (m,1H, NCH₂C═CH₂CH₂), 0.90 (s, 9H, SiC(CH₃)₃), 0.06 (s, 6H, Si(CH₃)₂); ¹³CNMR (67.8 MHz, CDCl₃) δ150.0 (NCH₂C═CH₂), 104.7 (NCH₂C═CH₂), 64.7(NCHCH₂OTBDMS), 60.5 (NCHCH₂OTBDMS), 51.3 (NCH₂C═CH₂), 34.9(NCH₂C═CH₂CH₂), 25.9 (SiC(CH₃)₃), 18.3 (SiC(CH₃)₃), −5.4 (Si(CH3)₂); MS(EI), m/z (relative intensity) 227 (M^(+.), 8), 212 (6), 170 (M—^(t)Bu,36), 96 (8), 82 (M—CH₂OTBDMS, 100), 75 (11); IR (Neat) 3550-3100 (br,NH), 3074 (C═CH₁₂), 2929, 2857, 1664 (C═C), 1472, 1424, 1391, 1380,1361, 1255, 1190, 1101, 1006, 939, 880, 838, 777, 723, 668 cm⁻¹.

(2S)-N-[5-Iodo-2-(2,2,2-trichloroethyloxycarbonylamino)benzoyl]-2-(tert-butyldimethylsilyloxymethyl)-4-methylidinepyrrolidine(60)

A catalytic amount of DMF (3 drops) was added to a stirred solution ofthe Troc protected anthranilic acid 59 (0.46 g, 1.04 mmol) and oxalylchloride (0.10 mL, 0.15 g, 1.15 mmol) in CH₂Cl₂ (30 mL). After 16 hoursat room temperature the resulting acid chloride solution was addeddropwise over 30 minutes to a stirred mixture of the amine 58 (0.26 g,1.15 mmol) and TEA (0.26 g, 0.36 mL, 2.58 mmol) in CH₂Cl₂ (15 mL) at−20° C. (CCl₄/liq.N₂) under a nitrogen atmosphere. The reaction mixturewas allowed to warm to room temperature and stirred for a further 45minutes. At this point TLC analysis (50% EtOAc/Petroleum Ether) revealedcomplete reaction. The mixture was washed with saturated NaHCO₃ (30 mL),saturated NH₄Cl (30 mL), H₂O (25 mL), brine (30 mL), dried (MgSO₄),filtered and evaporated in vacuo to give the amide 60 as a dark oil(0.65 g, 96%): ¹H NMR (270 MHz, CDCl₃) (Rotamers) δ8.92 (br s, 1H),8.05-7.88 (m, 1H), 7.74-7.64 (m, 1H), 7.56-7.46 (m, 1H), 5.08-4.95 (m,2H), 4.84 (d, 1H, J=11.91 Hz), 4.75 (d, 1H, J=11.91 Hz), 4.74-4.65 (m,1H), 4.21-3.68 (m, 4H), 2.96-2.65 (m, 2H), 0.95-0.87 (m, 9H), 0.1-0.03(m, 6H).

(2S)-N-(2-Amino-5-iodobenzoyl)-2-(hydroxymethyl)-4-methylidenepyrrolidine(61)

A solution of TBAF (1.24 mL of a 1M solution in THF, 1.24 mmol) wasadded to the silyl-ether 60 (0.64 g, 0.99 mmol) in THF (15 mL) at 0° C.(ice/acetone). The reaction mixture was allowed to warm to roomtemperature and after 45 minutes TLC (50% EtOAc/Pet-Ether 40°-60°)revealed the complete disappearance of starting material. SaturatedNH₄Cl (75 mL) was added and the reaction mixture extracted with EtOAc(3×30 mL), washed with brine (30 mL), dried (MgSO₄), filtered andevaporated in vacuo to give an orange oil. Purification by flashchromatography (50% EtOAc/Pet-Ether 40°-60°) provided the pureamino-alcohol 61 as a viscous oil (0.18 g, 51%): ¹H NMR (270 MHz, CDCl₃)δ7.72-7.61 (m, 1H), 7.55-7.40 (m, 1H), 6.51-6.49 (m, 1H), 5.02-4.94 (m,2H), 4.80-3.80 (m, 8H), 2.81-2.79 (m, 1H), 2.43-2.40 (m, 1H); MS (EI),m/z (relative intensity) 359 (M^(+.)1, 5), 358 (M^(+.), 33), 328 (3),327 (10), 254 (3), 247 (11), 246 (100), 218 (18), 164 (2), 127 (4), 120(4), 119 (10), 113 (9), 112 (91), 94 (2), 91 (20), 90 (5), 82 (10), 67(2), 64 (3), 63 (3), 52 (3). ps(2S)-N-[5-Iodo-2-(2,2,2-trichloroethyloxycarbonylamino)benzoyl]-2-(hydroxymethyl)-4-methylidinepyrrolidine(62)

A solution of the amine 61 (179 mg, 0.50 mmol) in CH₂Cl₂ (15 mL) wascooled to 0° C. (ice/acetone) and treated with pyridine (81 μL, 79 mg,1.0 mmol). A solution of 2,2,2-trichloroethylchloroformate (76 μL, 117mg, 0.55 mmol) in CH₂Cl₂ (5 mL) was then added dropwise to the stirredmixture. The reaction mixture was allowed to warm to room temperatureand stirred for a further 2 h, at which point TLC (EtOAc) revealedcomplete consumption of amine 61. The reaction mixture was washed withsaturated CuSO₄ (25 mL), H₂O (25 mL), brine (25 mL), dried (MgSO₄),filtered and evaporated in vacuo. The crude residue was purified byflash chromatography (50% EtOAc/Petroleum Ether) to afford the puretroc-amino compound 62 as an oil (189 mg, 71%): ¹H NMR (270 MHz, CDCl₃)δ8.90 (br s, 1H), 7.75-7.66 (m, 3H), 5.02-4.92 (m, 3H), 4.87 (d, 1H,J=12.09 Hz), 4.72 (d, 1H, J=12.09 Hz), 4.15-4.08 (m, 2H), 3.90-3.85 (m,2H), 3.65-3.63 (m, 1H), 2.80-2.71 (m, 1H), 2.50 (d, 1H, J=14.83 Hz); ¹³CNMR (67.8 MHz, CDCl₃) δ167.7, 151.9, 142.7, 139.6, 135.6, 134.8, 127.7,123.4, 108.4, 95.1, 86.6, 74.3, 63.9, 59.0, 53.5, 33.7; MS (EI), m/z(relative intensity) 536 (5), 535 (3), 534 (15), 533 (M^(+.), 3), 532(15), 503 (2), 501 (2), 422 (4), 420 (5), 385 (8), 384 (8), 366 (3), 353(11), 290 (9), 273 (8), 272 (76), 246 (6), 245 (18), 218 (4), 217 (5),216 (8), 146 (4), 145 (10), 133 (4), 131 (4), 119 (6), 117 (7), 115(11), 113 (17), 112 (39), 97 (4), 96 (3), 95 (12), 90 (5), 84 (5), 83(7), 82 (100), 79 (7), 77 (21), 67 (2), 63 (4), 61 (3), 51 (6); exactmass calcd for C₁₆H₁₁N₂O₄Cl₃I m/e 531.9221, obsd m/e 531.9155.

(11S,11aS)-11-Hydroxy-7-iodo-2-methylidene-10-(2,2,2-trichloroethyloxycarbonylamino)-1,2,3,10,11,1la-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(63)

A solution of the alcohol 62 (189 mg, 0.35 mmol) in CH₂Cl₂/CH₃CN (12 mL,3:1) was treated with 4 A powdered molecular sieves (100 mg) and NMO (62mg, 0.53 mmol). After 15 minutes stirring at room temperature, TPAP (6.2mg, 17.7 Amos) was added and stirring continued for a further 1 hour atwhich point TLC (50% EtOAc/Petroleum Ether) showed product formationalong with some unoxidised starting material. The mixture was thentreated with a further quantity of NMO (62 mg, 0.53 mmol) and TPAP (6.2mg, 17.7 μmol) and allowed to stir for a further 30 minutes after whichtime TLC revealed complete reaction. The mixture was evaporated in vacuoonto silica and subjected to flash chromatography (40% EtOAc/PetroleumEther) to provide the protected carbinolamine 63 as a white glass (93mg, 49%): ¹H NMR (270 MHz, CDCl₃) δ8.09 (d, 1H, J=2.01 Hz), 7.80 (dd,1H, J=8.43, 2.20 Hz), 7.10 (d, 1H, J=8.43 Hz), 5.60 (d, 1H, J=9.71 Hz),5.20-5.04 (m, 3H), 4.79-4.50 (m, 1H), 4.32-4.08 (m, 3H), 3.63 (t, 1H,J=8.79 Hz), 2.99-2.89 (m, 1H), 2.72 (d, 1H, J=15.94 Hz); ¹³C NMR (67.8MHz, CDCl₃) δ165.0, 154.1, 141.0, 140.2, 137.7, 134.5, 133.6, 132.0,110.4, 94.7, 93.4, 85.7, 75.0, 59.4, 50.7, 35.0; MS (EI), m/z (relativeintensity) 533 (6), 532 (22), 531 (M^(+.), 8), 530 (17), 529 (10), 449(5), 383 (6), 354 (7), 353 (5), 338 (6), 325 (5), 290 (5), 274 (15), 273(8), 272 (43), 254 (5), 245 (8), 218 (5), 216 (12), 147 (5), 146 (6),145 (9), 133 (10), 131 (9), 128 (5), 127 (15), 119 (11), 117 (5), 97(6), 95 (9), 92 (6), 91 (6), 90 (6), 83 (11), 82 (100), 81 (7), 80 (8),75 (5), 63 (7), 53 (5); exact mass calcd for C₁₆H₁₄N₂O₄ICl₃ m/e531.9037, obsd m/e 531.8988.

(11aS)-7-Iodo-2-methylidene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(64, UP2023, BSD-SJG)

10% cadmium-lead couple (109 mg, 0.875 mmol) was added to a stirredsolution of the Troc-protected carbinolamine 63 (93 mg, 0.175 mmol) inTHF (1 mL) and aqueous IN ammonium acetate (1 mL). After 45 minutes atroom temperature TLC revealed complete reaction (70% EtOAc/PetroleumEther). The mixture was diluted with EtOAc (30 mL), dried (MgSO₄),filtered and evaporated in vacuo. The crude residue was purified byflash chromatography (70% EtOAc/Petroleum Ether) to provide the novelPBD (64, BSD-SJG, UP2023) as a white solid (27 mg, 46%): mp ° C.; ¹H NMR(270 MHz, CDCl₃+CD₃OD) (11S,11aS isomer) δ8.10 (d, 1H, J=1.46 Hz), 7.65(d, 1H, J=8.79 Hz), 6.86 (d, 1H, J=8.06 Hz), 5.14-5.10 (m, 2H), 4.66 (d,1H, J=5.13 Hz), 4.34 (d, 1H, J=16.12 Hz), 4.23 (d, 1H, J=16.12 Hz),3.80-3.71 (m, 1H), 3.34 (s, 3H), 3.03-2.86 (m, 1H), 2.65 (d, 1H, J=16.02Hz); MS (ET), m/z (relative intensity) (N10-C11 imine form) 339 (M^(+.),+1, 20), 338 (M^(+.), 100), 337 (17), 323 (5), 311 (4), 310 (5), 257(5), 230 (4), 229 (13), 211 (4), 203 (4), 202 (8), 184 (8), 183 (4), 103(5), 82 (17), 81 (4), 80 (5), 76 (6), 75 (16), 74 (5), 55 (4), 53 (4);IR (NUJOL®) 3295 (br), 2923, 2853, 1716, 1615, 1506, 1457, 1377, 1317,1278, 1238, 1169, 1118, 1063, 999, 895, 818, 751, 718 cm⁻¹; exact masscalcd for C₁₃H₁₁N₂OI m/e 337.9916, obsd m/e 337.9870.

Example 2(b)

Synthesis of the C8-Benzyl-C7-Methoxy-C2-methlene PBD Monomer SJG-244(70) (See FIG. 8)

(2S)-N-(4-Benzyloxy-5-methoxy-2-nitrobenzoyl)-2-(tert-butyldimethylsilyloxymethyl)-4-methylidenepyrrolidine(65)

A catalytic amount of DMF (2 drops) was added to a stirred solution ofthe nitro-acid 1 (0.645 g, 2.13 mmol) and oxalyl chloride (0.23 mL, 0.33g, 2.60 mmol) in CH₂Cl₂ (40 mL). After 16 hours at room temperature theresulting acid chloride solution was added dropwise to a stirred mixtureof the amine 58 (0.522 g, 2.30 mmol) and TEA (0.58 g, 0.80 mL, 5.73mmol) in CH₂Cl₂ (5 mL) at 0° C. (ice/acetone) under a nitrogenatmosphere. The reaction mixture was allowed to warm to room temperatureand stirred for a further 2.5 h. The mixture was diluted with CH₂Cl₂ (50mL), washed with saturated NaHCO₃ (50 mL), saturated NH₄Cl (50 mL), H₂O(50 mL), brine (50 mL), dried (MgSO₄), filtered and evaporated in vacuoto give the crude product as a dark orange oil. Purification by flashchromatography (20% EtOAc/Petroleum Ether) isolated the pure amide 65 asa sticky orange oil (0.86 g, 79%): [α]²² _(D)=−47.2° (c=2.79, CHCl₃); ¹HNMR (270 MHz, CDCl₃) (Rotamers) δ7.78 and 7.77 (s×2, 1H_(arom)),7.48-7.35 (m, 5H_(arom)), 6.82 and 6.78 (s×2, 1H_(arom)), 5.23 and 5.21(s×2, 2H, PhCH₂O), 5.09-4.83 (m, 2H, NCH₂C═CH₂), 4.59-4.49 (m, 1H,NCHCH₂OTBDMS), 4.03-3.08 (m, 7H, NCHCH₂OTBDMS, NCH₂C═CH₂ and OCH₃),2.80-2.56 (m, 2H, NCH₂C═CH₂CH₂), 0.89 and 0.79 (s×2, 9H, SiC(CH₃)₃),0.122, −0.11 and −0.14 (s×3, 6H, Si(CH₃)₂); ¹³C NMR (67.8 MHz, CDCl₃)(Rotamers) δ166.2 (NC═O), 154.8 and 154.6 (C_(quat)), 148.2 and 148.0(C_(quat)), 144.1 and 143.2 (C_(quat)), 137.1 (C_(quat)), 135.3(C_(quat)), 128.8 and 128.5 (BnC—H_(arom)), 128.2 (C_(quat)), 127.6(BnC-H_(arom)), 110.1 and 109.2 (C—H_(arom)), 109.0 and 108.5(C—H_(arom)), 107.5 (NCH₂C═CH₂), 71.3 (PhCH₂O), 63.7 (NCHCH₂OTBDMS),60.2 (NCHCH₂OTBDMS), 58.1 and 56.6 (OCH3), 52.8 and 50.5 (NCH₂C₂═CH₂),34.9 and 33.9 (NCH₂C═CH₂CH₂), 25.8 and 25.7 (SiC(CH₃)₃), 18.2(SiC(CH₃)₃), −5.4 and −5.6 (Si(CH₃)₂); MS (EI), m/z (relative intensity)512 (M^(+.), 3), 497 (M—CH₃, 4), 455 (M—^(t)Bu, 100), 380 (2), 364 (5),286 (M—NCH₂C═CH₂CH₂CHCH₂OTBDMS, 40), 279 (9), 226(NCH₂C═CH₂CH₂CHCH₂OTBDMS, 5), 168 (10), 149 (27), 91 (PhCH₂, 62), 73(8), 57 (9); IR (NEAT) 3066, 3034, 2953, 2856, 2245, 1644 (NC═O), 1578,1520, 1454, 1426, 1379, 1335, 1276, 1220, 1186, 1106, 1059, 1016, 910,836, 815, 779, 734, 697, 655, 614 cm¹.

(2S)-N-(4-Benzyloxy-5-methoxy-2-nitrobenzoyl)-2-(hydroxymethyl)-4-methylidenepyrrolidine(66)

A solution of TBAF (2.10 mL of a 1M solution in THF, 2.10 mmol) wasadded to the silyl-ether 65 (0.86 g, 1.68 mmol) in THF (20 mL) at 0° C.(ice/acetone). The reaction mixture was allowed to warm to roomtemperature following a colour change (yellow-dark red). After a further40 minutes TLC (50% EtOAc/Pet-Ether 40°-60°) revealed the completedisappearance of starting material. Saturated NH₄Cl (100 mL) was addedand the reaction mixture extracted with EtOAc (3×40 mL), washed withbrine (30 mL), dried (MgSO₄), filtered and evaporated in vacuo to give adark orange oil which was purified by flash chromatography (60%EtOAc/Petroleum Ether) to provide the pure alcohol 66 as a white solid(0.64 g, 96%): [α]¹⁹ _(D)=−22.9° (c=0.20, MeOH); ¹H NMR (270 MHz, CDCl₃)(Rotamers) δ7.78 and 7.76 (s×2, 1H_(arom)), 7.49-7.33 (m, 5H_(arom)),6.91 and 6.82 (s×2, 1H_(arom)), 5.22 (s, 2H, PhCH₂O), 5.10 (m, 1H, OH),5.03-5.01 (m, 1H, NCH₂C═CH₂), 4.90-4.85 (m, 1H, NCH₂C═CH₂), 4.65-4.55(m, 1H, NCHCH₂OH), 3.99 and 3.95 (s×2, 3H, OCH₃), 3.90-3.72 (m, 4H,NCHCH₂OH and NCH₂C═CH₂), 2.90-2.87 (m, 1H, NCH₂C═CH₂CH₂), 2.53-2.47 (m,1H, NCH₂C═CH₂CH₂); ¹³C NMR (67.8 MHz, CDCl₃) (Rotamers) δ177.4 (NC═O),155.1 (C_(quat)), 148.3 (C_(quat)), 142.6 (C_(quat)), 137.0 (C_(quat)),135.2 (C_(quat)), 128.9, 128.6 and 127.6 (BnC—H_(arom)), 109.1(C—H_(arom)), 108.5 (C—H_(arom)), 108.3 (NCH₂C═CH₂), 71.4 (PhCH₂O), 65.2and 63.7 (NCHCH₂OH), 60.4 (NCHCH₂OH), 56.8 and 56.7 (OCH3), 53.0 and50.1 (NCH₂C═CH₂), 35.1 and 34.4 (NCH₂C═CH₂CH₂); MS (EI), m/z (relativeintensity) 398 (M^(+.), 2), 380 (3), 368 (4), 354 (1), 286(M—NCH₂C═CH₂CH₂CHCH₂OH, 54), 270 (2), 256 (1), 164 (2), 136 (4), 135(3), 121 (4), 112 (NCH₂C═CH₂CH₂CHCH₂OH, 3), 91 (PhCH₂, 100), 82 (3), 69(4), 65 (6); IR (NUJOL®) 3600-3200 (br, OH), 2923, 2853, 1718, 1663,1611 (NC═O), 1577, 1517, 1460, 1376, 1332, 1275, 1224, 1176, 1052, 990,925, 886, 862, 796, 759, 723, 702 615 cm⁻¹; exact mass calcd forC₂₁H₂₂N₂O₆ m/e 398.1478, obsd m/e 398.1490.

(2S)-N-(2-Amino-4-benzyloxy-5-methoxybenzoyl)-2-(hydroxymethyl)-4-methylidenepyrrolidine(67)

The nitro-alcohol 66 (0.637 g, 1.60 mmol), SnCl₂ 2H₂O (1.81 g, 8.0 mmol)and methanol (36 mL) were heated at reflux and monitored by TLC (90%CHCl₃/MeOH). After 1 hour the MeOH was evaporated in vacuo and theresulting residue cooled (ice), and treated carefully with saturatedNaHCO₃ (120 mL). The mixture was diluted with EtOAc (120 mL), and after16 hours stirring at room temperature the inorganic precipitate wasremoved by filtration through celite. The organic layer was separated,washed with brine (100 mL), dried (MgSO₄), filtered and evaporated invacuo to give an orange glass. Flash chromatography (EtOAc) afforded thepure amine 67 as a pale yellow glass (0.37 g, 63%): [α]²³ _(D)=−42.7°(c=3.7, CHCl₃); ¹H NMR (270 MHz, CDCl₃) δ7.44-7.29 (m, 5H_(arom)), 6.77(s, 1H_(arom)) 6.27 (s, 1H_(arom)), 5.12 (s, 2H, PhCH₂O), 5.06-5.00 (m,1H, NCH₂C═CH₂), 4.99-4.92 (m, 1H, NCH₂C═CH₂), 4.63-4.53 (m, 1H,NCHCH₂OH), 4.25-3.60 (m, 10H, NCHCH₂OH, NCH₂C═CH₂, OCH₃, OH and NH₂),2.77 (dd, 1H, J=8.52, 15.85 Hz, NCH₂C═CH₂CH₂), 2.43-2.39 (m, 1H,NCH₂C═CH₂CH₂); ¹³C NMR (67.8 MHz, CDCl₃) δ171.4 (NC═O), 151.0(C_(quat)), 143.3 (C_(quat)), 141.5 (C_(quat)), 140.6 (C_(quat)), 136.5(C_(quat)), 128.6 and 128.0 (BnC—H_(arom)), 127.8 (C_(quat)), 127.1(BnC—H_(arom)), 112.5 (C—H_(arom)), 107.8 (NCH₂C═CH₂), 103.0(C—H_(arom)), 70.6 (PhCH₂O), 65.9 (NCHCH₂OH), 60.0 (NCHCH₂OH), 57.1(OCH₃), 53.3 (NCH₂C═CH₂), 34.4 (NCH₂C═CH₂CH₂); MS (EI), m/z (relativeintensity) 368 (M^(+.), 100), 353 (M—CH₃, 2), 340 (1), 286 (2), 273 (4),256 (M—NCH₂C═CH₂CH₂CHCH₂OH, 59), 249 (8), 226 (4), 200 (2), 196 (2), 166(5), 138 (17), 112 (NCH₂C═CH₂CH₂CHCH₂OH, 39), 91 (PhCH₂, 70), 82 (5), 65(5); IR (NEAT) 3600-3000 (br, NH₂ and OH), 3065, 3052, 2932, 2869, 2246,1668, 1620, 1592, 1513, 1454, 1408, 1264, 1229, 1197, 1176, 1113, 1079,1002, 909, 733, 698, 645 cm⁻¹; exact mass calcd for C₂₁H₂₄N₂O₄ m/e368.1736, obsd m/e 368.1662.

(2S)-N-[(2-Allyloxycarbonylamino)-4-benzyloxy-5-methoxybenzoyl]-2-(hydroxymethyl)-4-methylidenepyrrolidine(68)

A solution of the amino-alcohol 67 (0.33 g, 0.90 mmol) in CH₂Cl₂ (20 mL)was cooled to 0° C. (ice/acetone) and treated with pyridine (0.14 mL,0.14 g, 1.77 mmol). A solution of allyl chloroformate (87 μL, 99 mg,0.82 mmol) in CH₂Cl₂ (7 mL) was then added dropwise to the stirredmixture. The reaction mixture was allowed to warm to room temperatureand stirred for a further 2.5 h, at which point TLC (EtOAc) revealedcomplete consumption of amine 67. The reaction mixture was diluted withCH₂Cl₂ (30 mL) and washed with saturated CuSO₄ (40 mL), H₂O (40 mL),brine (40 mL), dried (MgSO₄), filtered and evaporated in vacuo. Thecrude residue was purified by flash chromatography (80% EtOAc/PetroleumEther) to afford the pure alloc-amino compound 68 as a white solid (0.34g, 84%): [α]²² _(D)=22.4° (c=3.4, CHCl₃); ¹H NMR (270 MHz, CDCl₃) δ8.52(br s, 1H, NH), 7.82 (br s, 1H_(arom)), 7.49-7.29 (m, 5H_(arom)), 6.84(s, 1H_(arom)), 6.02-5.88 (m, 1H, NCO₂CH₂CH═CH₂), 5.39-5.22 (m, 2H,NCO₂CH₂CH═CH₂), 5.17 (s, 2H, PhCH₂O), 5.01 (br s, 1H, NCH₂C═CH₂), 4.94(br s, 1H, NCH₂C═CH₂), 4.64-4.59 (m, 3H, NCHCH₂OH and NCO₂CH₂CH═CH₂),4.21-3.60 (m, 8H, NCHCH₂OH, NCH₂C═CH₂, OCH₃ and OH), 2.77 (dd, 1H,J=8.61, 15.94 Hz, NCH₂C═CH₂CH₂), 2.46 (d, 1H, J=15.94 Hz, NCH₂C═CH₂CH₂);¹³C NMR (67.8 MHz, CDCl₃) δ171.4 (NC═O_(amide)), 153.7(NC=O_(carbamate)), 150.3 (C_(quat)), 144.5 (C_(quat)), 143.0(C_(quat)), 136.2 (C_(quat)), 132.4 (NCO₂CH₂CH═CH₂), 131.3 (C_(quat)),128.6, 128.1, and 127.7 (BnC—H_(arom)), 118.1 (NCO₂CH₂CH═CH₂), 111.1(C—H_(arom)), 108.1 (NCH₂C═CH₂), 106.5 (C—H_(arom)), 70.7 (PhCH₂O), 65.8(NCO₂CH₂CH═CH₂), 65.5 (NCHCH₂OH), 59.9 (NCHCH₂OH), 56.7 (OCH₃), 54.0(NCH₂C═CH₂), 34.1 (NCH₂C═CH₂CH₂); MS (EI), m/z (relative intensity) 452(M^(+.), 38), 395 (M—OC₃H₅, 4), 394 (10), 340 (M—NCH₂C═CH₂CH₂CHCH₂OH,20), 298 (7), 282 (22), 255 (8), 206 (2), 192 (2), 163 (3), 136 (3), 114(6), 112 (NCH₂C═CH₂CH₂CHCH₂OH, 12), 91 (PhCH₂, 100), 82 (10), 65 (4), 57(OC₃H₅, 7); IR (NUJOL®) 3600-2000 (br, OH), 3335, 3242, 2922, 2854,1724, 1614, 1537, 1463, 1407, 1378, 1349, 1280, 1214, 1178, 1117, 1054,1028, 995, 947, 908, 892, 853, 821, 768, 735, 697, 629, 601, 514 cm⁻¹;exact mass calcd for C₂₅H₂₈N₂O₆ m/e 452.1947, obsd m/e 452.1923.

(11S,11aS)-10-Allyloxycarbonyl-8-benzyloxy-11-hydroxy-7-methoxy-2-methylidene-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(69)

A solution of DMSO (0.18 mL, 0.20 g, 2.56 mmol) in CH₂Cl₂ (4 mL) wasadded dropwise over 30 minutes to a solution of oxalyl chloride (0.63 mLof a 2.0 M solution in CH₂Cl₂, 1.26 mmol) at −45° C. (dry ice/CH₃CN)under a nitrogen atmosphere. After stirring at −45° C. for 30 minutes, asolution of the alcohol 68 (0.42 g, 0.93 mmol) dissolved in CH₂Cl₂ (8mL) was added dropwise over 35 minutes at −45° C. After 45 minutes at−45° C., the mixture was treated dropwise with TEA (0.50 mL, 0.36 g,3.56 mmol) in CH₂Cl₂ (4 mL) over 30 minutes at −45° C. After 35 minutes,the reaction mixture was allowed to warm to room temperature and wasdiluted with CH₂Cl₂ (30 mL), washed with 1N HCl (20 mL), H₂O (20 mL),brine (30 mL), dried (MgSO₄), filtered and evaporated in vacuo. TLC (80%EtOAc/Petroleum Ether) of the crude material revealed sufficient productformation and a trace of unoxidised starting material. Purification byflash chromatography (50% EtOAc/Petroleum Ether) furnished the protectedcarbinolamine 69 as white glass (0.172 g, 41%): ¹H NMR (270 MHz, CDCl₃)δ7.48-7.27 (m, 5H_(arom)) 7.25 (s, 1H_(arom)), 6.74 (br s, 1H_(arom)),5.65-5.53 (m, 1H, NCO₂CH₂CH═CH₂), 5.56 (d, 1H, J=9.89 Hz, NCHCHOH),5.22-5.04 (m, 6H, NCH₂C═CH₂, NCO₂CH₂CH═CH₂ and PhCH₂O), 4.64-4.42 (m,3H, NCO₂CH₂CH═CH₂ and OH), 4.28 (d, 1H, J=15.94 Hz, NCH₂C═CH₂), 4.09 (d,1H, J=15.94 Hz, NCH₂C═CH₂), 3.92 (s, 3H, OCH₁₃), 3.62 (t, 1H, J=8.79 Hz,NCHCHOH), 2.90 (dd, 1H, J=8.97, 16.03 Hz, NCH₂C═CH₂CH₂), 2.67 (d, 1H,J=16.03 Hz, NCH₂C═CH₂CH₂); ¹³C NMR (67.8 MHz, CDCl₃) δ166.8(NC═O_(amide)), 156.0 (NC═O_(carbamate)), 150.1 (C_(quat)), 149. 0(C_(quat)), 141.8 (C_(quat)), 136. 1 (C_(quat)), 131.8 (NCO₂CH₂CH═CH₂,),128.6, 128.1 and 127.3 (BnC—H_(arom)), 125.6 (C_(quat)), 118.0(NCO₂CH₂CH═CH₂), 14.6 (C—H_(arom)), 110.6 (C—H_(arom)), 109.8(NCH₂C═CH₂), 85.8 (NCHCHOH), 71.0 (PhCH₂O), 66.7 (NCO₂CH₂CH═CH₂), 59.8(NCHCHOH), 56.2 (OCH3), 50.7 (NCH₂C═CH₂), 35.0 (NCH₂C═CH₂CH₂); MS (EI),m/z (relative intensity) 450 (M^(+.), 24), 422 (1), 392 (1), 364 (1),348 (3), 340 (12), 298 (6), 282 (8), 257 (2), 229 (2), 192 (3), 178 (2),164 (4), 136 (3), 110 (3), 91 (PhCH₂, 100), 82 (17), 65 (7); IR (NUJOL™)3600-2500 (br, OH), 2923, 2854, 1711, 1619, 1601, 1513, 1463, 1405,1377, 1300, 1278, 1202, 1119, 1045, 993, 956, 909, 790, 768, 724, 697,637 cm⁻¹; exact mass calcd for C₂₅H₂₆N₂O₆ m/e 450.1791, obsd m/e450.1790.

Alternative Synthesis(11S,11aS)-10-Allyloxycarbonyl-8-benzyloxy-11-hydroxy-7-methoxy-2-methylidene-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(69)

A solution of the alcohol 68 (0.32 g, 0.71 mmol) in CH₂Cl₂/CH₃CN (30 mL,3:1) was treated with 4 A powdered molecular sieves (0.2 g) and NMO (126mg, 1.08 mmol). After 15 minutes stirring at room temperature, TPAP(12.6 mg, 35.9 μmol) was added and stirring continued for a further 1hour 20 minutes at which point TLC (80% EtOAc/Petroleum Ether) revealedproduct formation along with some unoxidised starting material. Themixture was then treated with a further quantity of NMO (126 mg, 1.08mmol) and TPAP (12.6 mg, 35.9 μmol), and allowed to stir for a further0.5 hours after which time TLC revealed reaction completion. The mixturewas evaporated in vacuo onto silica and subjected to flashchromatography (50% EtOAc/Petroleum Ether) to provide the protectedcarbinolamine 69 as a white glass (153 mg, 48%): [α]²³ _(D)=+129.8°(c=1.5, CHCl₃); ¹H NMR (270 MHz, CDCl₃) δ7.48-7.27 (m, 5H_(arom)), 7.25(s, 1H_(arom)), 6.74 (br s, 1H_(arom)), 5.65-5.53 (m, 1H,NCO₂CH₂CH═CH₂), 5.56 (d, 1H, J=9.89 Hz, NCHCHOH), 5.22-5.04 (m, 6H,NCH₂C═CH₂, NCO₂CH₂CH═CH₂ and PhCH₂O), 4.64-4.42 (m, 3H, NCO₂CH2CH═CH₂and OH), 4.28 (d, 1H, J=15.94 Hz, NCH₂C═CH₂), 4.09 (d, 1H, J=15.94 Hz,NCH₂C═CH₂), 3.92 (s, 3H, OCH₃), 3.62 (t, 1H, J=8.79 Hz, NCHCHOH), 2.90(dd, 1H, J=8.97, 16.03 Hz, NCH₂C═CH₂CH₂), 2.67 (d, 1H, J=16.03 Hz,NCH₂C═CH₂CH₂); ¹³C NMR (67.8 MHz, CDCl₃) δ166.8 (NC═O_(amide)), 156.0(NC═O_(carbamate)), 150.1 (C_(quat)), 149.0 (C_(quat)), 141.8(C_(quat)), 136.1 (C_(quat)), 131.8 (NCO₂CH₂CH═CH₂), 128.6, 128.1 and127.3 (BnC—H_(arom)), 125.6 (C_(quat)), 118.0 (NCO₂CH₂CH═CH₂), 114.6(C—H_(arom)), 110.6 (C—H_(arom)), 109.8 (NCH₂C═CH₂), 85.8 (NCHCHOH),71.0 (PhCH₂O), 66.7 (NCO₂CH₂CH═CH₂), 59.8 (NCHCHOH), 56.2 (OCH₃), 50.7(NCH₂C═CH₂), 35.0 (NCH₂C═CH₂CH₂); MS (EI), m/z (relative intensity) 450(M^(+.) , 24), 422 (1), 392 (1), 364 (1), 348 (3), 340 (12), 298 (6),282 (8), 257 (2), 229 (2), 192 (3), 178 (2), 164 (4), 136 (3), 110 (3),91 (PhCH₂, 100), 82 (17), 65 (7); IR (NUJOL®) 3600-2500 (br, OH), 2923,2854, 1711, 1619, 1601, 1513, 1463, 1405, 1377, 1300, 1278, 1202, 1119,1045, 993, 956, 909, 790, 768, 724, 697, 637 cm⁻¹; exact mass calcd forC₂₅H₂₆N₂O₆m/e 450.1791, obsd ml/e 450.1790.

(11aS)-8-Benzyloxy-7-methoxy-2-methylidene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(70, SJG-244)

A catalytic amount of tetrakis(triphenylphosphine)palladium (12.0 mg,10.4 μmol) was added to a stirred solution of the Alloc-protectedcarbinolamine 69 (0.18 g, 0.40 mmol), triphenylphosphine (5.25 mg, 20μmol) and pyrrolidine (29 mg, 0.41 mmol) in CH₂Cl₂ (15 mL). After 2hours stirring at room temperature under a nitrogen atmosphere, TLC (98%CHCl₃/MeOH) revealed the complete consumption of starting material. Thesolvent was evaporated in vacuo and the crude residue was purified byflash chromatography (60% EtOAc/Petroleum Ether) to afford 70 (SJG-244)as a white glass (116 mg, 83%) which was repeatedly evaporated in vacuowith CHCl₃in an attempt to provide the N10-C11 imine form: [α]²²_(D)=+754.2° (c=0.54, CHCl₃); ¹H NMR (270 MHz, CDCl₃) (mainly imine,plus trace of carbinolamine form) δ7.70-7.30 (m, 7H, HC═N and6H_(arom)), 6.84 (s, 1H_(arom)), 5.25-5.13 (m, 4H, NCH₂C═CH₂ andPhCH₂O), 4.42 (br s, 2H, NCH₂C═CH₂), 3.95 (s, 3H, OCH₃), 3.88-3.66 (m,1H, NCHHC═N), 3.09 (dd, 1H, J 8.98, 16.12 Hz, NCH₂C═CH₂CH₂), 2.94-2.87(m, 1H, NCH₂C═CH₂CH₂); ¹³C NMR (67.8 MHz, CDCl₃) δ164.7 (NC═O), 162.6(HC═N), 150.6 (C_(quat)), 148.1 (C_(quat)), 141.6 (C_(quat)), 140.5(C_(quat)), 136.1 (C_(quat)), 132.0, 128.7, 128.6, 128.1 and 127.3(BnC—H_(arom)), 120.1 (C_(quat)), 111.5 (C—H_(arom)), 111.2(C—H_(arom)), 109.4 (NCH₂C═CH₂), 70.8 (PhCH₂O), 56.2 (OCH₃), 53.7(NCHHC═N), 51.3 (NCH₂C═CH₂), 35.4 (NCH₂C═CH₂CH₂); MS (EI), m/z (relativeintensity) (imine form) 348 (M^(+.), 100), 333 (M—CH₃, 42), 319 (3), 269(5), 257 (M—PhCH₂, 25), 241 (11), 229 (56), 227 (11), 213 (5), 186 (4),156 (6), 136 (22), 122 (4), 91 (PhCH₂, 85), 82 (5), 65 (22); IR (NUJOL™)3318 (br, OH of carbinolamine form), 2923, 2853, 1722, 1668, 1600, 1557,1504, 1462, 1377, 1261, 1216, 1120, 1003, 892, 789, 722, 695, 623, 542cm⁻¹; exact mass calcd for C₂₁H₂₀N₂O₃ m/e 348.1474, obsd m/e 348.1469.

Example 2(c)

Synthesis of MMY-SJG (74, UP2064) (See FIG. 9)

(2s)-N-[(2-Allyloxycarbonylamino)-4,5-dimethoxybenzoyl]-2-(tert-butyldimethylsilyloxymethyl)-4-methylidinepyrrolidine(71)

Potassium tert-butoxide (21.2 mL of a 0.5 M solution in THF, 10.6 mmol)was added dropwise to a suspension of methyltriphenylphosphonium bromide(3.78 g, 10.6 mmol) in THF (11 mL) at 0° C. (ice/acetone) undernitrogen. After stirring for 2 hours at 0° C., a solution of the ketone38 (Example 1(e)) (2.0 g, 4.07 mmol) in THF (7 mL) was added dropwiseand the mixture allowed to warm to room temperature. After stirring fora further 45 minutes the reaction mixture was diluted with EtOAc (60 mL)and water (60 mL). The organic layer was separated, washed with brine,dried (MgSO₄), filtered and evaporated in vacuo to give a dark oil.Purification by flash chromatography (20% EtOAc/Petroleum Ether)isolated the pure olefin 71 as a transparent oil (1.71 g, 86%): [α]²²_(D)=−44.55° (c=0.20, CHCl₃); ¹H NMR (270 MHz, CDCl₃) (Rotamers) δ8.85(br s, 1H), 7.86 (s, 1H), 6.82 (s, 1H), 6.03-5.89 (m, 1H), 5.35 (ddd,1H, J=17.22, 3.11, 1.47 Hz), 5.24 (ddd, 1H, J=10.44, 2.75, 1.28 Hz),4.99-4.92 (m, 2H), 4.70-4.57 (m, 3H), 4.23-3.57 (m, 10H), 2.72-2.68 (m,2H), 0.96-0.85 (m, 9H), 0.09-0.03 (m, 6H); ¹³C NMR (67.8 MHz, CDCl₃)(Rotamers) δ168.7, 153.6, 150.9, 143.6, 132.5, 132.2, 118.1, 115.3,110.6, 107.1, 104.3, 65.7, 63.6, 56.3, 56.0, 33.1, 25.8, 18.1, −5.5 and−5.6; MS (EI), m/z (relative intensity) 492 (M^(+.) +2, 7), 491 (M^(+.)+1, 20), 490 (M^(+.), 50), 475 (4), 435 (10), 447 (3), 434 (29), 433(94), 376 (4), 375 (13), 348 (5), 333 (11), 332 (6), 294 (3), 265 (16),264 (100), 227 (8), 226 (24), 224 (5), 223 (18), 220 (15), 210 (4), 208(5), 207 (13), 206 (96), 192 (7), 180 (18), 179 (25), 170 (21), 169 (8),168 (28), 164 (13), 152 (7), 150 (13), 136 (10), 108 (5), 96 (5), 95(12), 94 (7), 89 (8), 82 (25), 75 (20), 73 (30), 59 (7), 58 (5), 57(41), 56 (7), 55 (4); IR (NEAT) 3324 (br, NH), 3082, 2953, 2930, 2857,1732, 1600, 1523, 1490, 1464, 1419, 1397, 1360, 1333, 1287, 1259, 1228,1203, 1172, 1115, 1039, 1004, 939, 837, 814, 777 666 cm⁻¹.

(2S)-N-[(2-Allyloxycarbonylamino)-4,5-dimethoxybenzoyl]-2-(hydroxymethyl)-4-methylidinepyrrolidine(72)

A solution of TBAF (4.29 mL of a 1M solution in THF, 4.29 mmol) wasadded to the silyl-ether 71 (1.68 g, 3.43 mmol) in THF (45 mL) at 0° C.(ice/acetone). The reaction mixture was allowed to warm to roomtemperature and after 1 hour TLC (50% EtOAc/Pet-Ether 40°-60°) revealedthe complete disappearance of starting material. Saturated NH₄Cl (110mL) was added and the reaction mixture extracted with EtOAc (3×50 mL),washed with brine (100 mL), dried (MgSO₄), filtered and evaporated invacuo to give a dark orange oil. Purification by flash chromatography(99% CHCl₃/MeOH) provided the pure alcohol 72 as a white solid (1.15 g,89%): [α]²¹ _(D)=−13.17° (c=0.15, CHCl₃); ¹H NMR (270 MHz, CDCl₃) δ8.59(br s, 1H), 7.69 (s, 1H), 6.82 (s, 1H), 6.03-5.89 (m, 1H), 5.35 (ddd,1H, J=17.22, 3.11, 1.65 Hz), 5.24 (ddd, 1H, J=10.44, 2.75, 1.28 Hz),5.02-4.94 (m, 2H), 4.66-4.62 (m, 3H), 4.23-3.57 (m, 11H), 2.77 (dd, 1H,J=15.94, 8.42 Hz), 2.48 (d, 1H, J=15.94 Hz); ¹³C NMR (67.8 MHz, CDCl)δ170.3, 153.8, 151.0, 144.2, 143.1, 132.5, 131.2, 118.1, 115.9, 110.4,108.1, 104.9, 65.8, 65.1, 59.8, 56.4, 56.0, 54.2, 34.1; MS (EI), m/z(relative intensity) 378 (M^(+.) +2, 3), 377 (M^(+.) +1, 14), 376(M^(+.), 51), 319 (3), 265 (10), 264 (62), 263 (4), 259 (8), 224 (4),223 (18), 220 (17), 208 (5), 207 (14), 206 (100), 192 (8), 190 (5), 180(27), 179 (29), 178 (4), 164 (23), 163 (4), 152 (12), 151 (6), 150 (19),137 (5), 136 (22), 135 (6), 125 (6), 120 (6), 113 (6), 112 (31), 109(6), 108 (11), 95 (4), 94 (9), 82 (28), 80 (8), 67 (5), 57 (5), 54 (7),53 (7); IR (NUJOL®) 3341 and 3245 (br, OH and NH), 3115, 2918, 2850,1727, 1616, 1540, 1464, 1399, 1378, 1351, 1283, 1264, 1205, 1179, 1117,1055, 1040, 996, 946, 909, 894, 855, 823, 768, 754, 722, 696, 623, 602cm⁻¹; exact mass calcd for C₁₁H₂₄N₂O₆ m/e 376.1634, obsd m/e 376.1614.

(11S,11aS)-10-Allyloxycarbonyl-7,8-dimethoxy-11-hydroxy-2-methylidene-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(73)

A solution of DMSO (0.75 mL, 0.82 g, 10.5 mmol) in CH₂Cl₂ (27 mL) wasadded dropwise over 38 minutes to a solution of oxalyl chloride (2.64 mLof a 2.0 M solution in CH₂Cl₂, 5.27 mmol) at −45° C.(liq.N₂/Chlorobenzene) under a nitrogen atmosphere. After stirring at−45° C. for 1 h, a solution of the alcohol 72 (1.10 g, 2.93 mmol) inCH₂Cl₂ (27 mL) was added dropwise over 1 hour at −45° C. After 1 hour at−45° C., the mixture was treated dropwise with a solution of TEA (1.71mL, 1.24 g, 12.29 mmol) in CH₂Cl₂ (15 mL) over 40 minutes at −45° C.After a further 30 minutes, the reaction mixture was allowed to warm toroom temperature and was diluted with CH₂Cl₂ (50 mL), washed with 1N HCl(50 mL), H₂O (50 mL), brine (50 mL), dried (MgSO₄), filtered andevaporated in vacuo. TLC (80% EtOAc/Petroleum Ether) of the crudematerial revealed reaction completion. Purification by flashchromatography (60% EtOAc/Petroleum Ether) furnished the protectedcarbinolamine 73 as a white glass (0.45 g, 41%): [α]²² _(D)=+236.51°(c=0.14, CHCl₃); ¹H NMR (270 MHz, CDCl₃) δ7.23 (s, 1H), 6.69 (s, 1H),5.83-5.81 (m, 1H), 5.60-5.58 (m, 1H), 5.34-5.23 (m, 4H), 4.74-4.66 (m,1H), 4.50-4.40 (m, 1H), 4.30 (d, 1H, J=15.94 Hz), 4.15 (d, 1H, J=15.93Hz), 3.96-3.86 (m, 7H), 3.65 (t, 1H, J=8.61 Hz), 2.92 (dd, 1H, 16.21,9.07 Hz), 2.70 (d, 1H, J=15.94 Hz); ¹³C NMR (67.8 MHz, CDCl₃) δ166.7,156.0, 150.8, 148.4, 141.8, 131.7, 128.5, 125.2, 118.1, 112.4, 110.3,109.8, 85.9, 66.8, 59.6, 56.3, 56.1, 50.7, 35.0; MS (EI), m/z (relativeintensity) 376 (M^(+.) +2, 6), 375 (Me^(+.)1, 22), 374 (M^(+.), 100),346 (5), 293 (8), 288 (10), 271 (5), 265 (11), 264 (67), 248 (5), 237(5), 223 (10), 220 (9), 209 (6), 208 (42), 207 (14), 206 (70), 192 (7),190 (5), 180 (17), 179 (16), 165 (8), 164 (15), 153 (5), 152 (10), 150(12), 149 (7), 137 (6), 136 (10), 135 (5), 125 (8), 110 (8), 108 (5), 94(5), 83 (5), 82 (59), 80 (7), ; IR (CHCl₃) 3275 (br, OH), 3075, 2936,2851, 1706, 1624, 1604, 1516, 1457, 1436, 1403, 1368, 1312, 1301, 1278,1262, 1218, 1119, 1074, 1045, 940, 916, 893, 867, 851, 666, 637 cm⁻¹;exact mass calcd for C₁₉H₂₂N₂O₆ m/e 374.1478, obsd m/e 374.1687.

(11aS)-7,8-Dimethoxy-2-methylidene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(74, UP2064, MMY-SJG)

A catalytic amount of tetrakis(triphenylphosphine)palladium (32.4 mg,28.1 μmol) was added to a stirred solution of the Alloc-protectedcarbinolamine 73 (0.42 g, 1.12 mmol), triphenylphosphine (14.7 mg, 56.2μmol) and pyrrolidine (83.9 mg, 1.18 mmol) in CH₂Cl₂ (55 mL). After 2.5hours stirring at room temperature under a nitrogen atmosphere, TLC (95%CHCl₃/MeOH) revealed the complete consumption of starting material. Thesolvent was evaporated in vacuo and the crude residue was purified byflash chromatography (CHCl₃) to afford the novel PBD (74, MMY-SJG,UP2064) as a yellow oil which was repeatedly evaporated in vacuo withCHCl₃ in order to provide the N10-C11 imine form (259 mg, 85%): [α]²²_(D)=+583.14° (c=1.42, CHCl₃); ¹H NMR (270 MHz, CDCl₃) δ7.69 (d, 1H,J=4.39 Hz), 7.51 (s, 1H), 6.82 (s, 1H), 5.21-5.17 (m, 2H), 4.44-4.23 (m,2H), 3.96-3.81 (m, 7H), 3.17-3.08 (m, 1H), 2.95 (d, 1H, J=14.29 Hz); ¹³CNMR (67.8 MHz, CDCl₃) δ164.7, 162.6, 151.5, 147.6, 141.6, 140.8, 119.8,111.2, 109.4, 109.4, 56.2, 56.1, 53.8, 51.4, 35.5; MS (EI), m/z(relative intensity) 273 (M^(+.) +1, 16), 272 (M^(+.), 100), 271 (35),270 (9), 255 (5), 243 (7), 241 (7), 230 (6), 228 (6), 226 (5), 212 (3),192 (4), 191 (16), 165 (4), 164 (19), 163 (4), 136 (22), 93 (6), 82 (7),80 (3), 53 (3); IR (NEAT) 3312 (br), 3083, 2936, 2843, 1624, 1603, 1505,1434, 1380, 1264, 1217, 1180, 1130, 1096, 1069, 1007, 935, 895, 837,786, 696, 666, 594, 542 cm⁻¹; exact mass calcd for C₁₁H₁₆N₂O₃ m/e272.1161, obsd m/e 272.1154.

Example 2(d)

Synthesis of the PBD Dimer SJG-136 (UP2001) (See FIG. 10)

(2S)-1,1′-[[(Propane-1,3-diyl)dioxy]bis[(2-nitro-5-methoxy-1,4-phenylene)carbonyl]]bis[2-(tert-butyldimethylsilyloxymethyl)-4-methylidenepyrrolidine](75)

A catalytic amount of DMF (2 drops) was added to a solution of the dimeracid 44 (0.66 g, 1.42 mmol) and oxalyl chloride (0.31 mL, 0.45 g, 3.55mmol) in THF (12 mL). The reaction mixture was stirred for 16 hoursunder nitrogen, concentrated in vacuo, and redissolved in THF (10 mL).The resulting solution of bis-acid chloride was added dropwise to theamine 58 (0.65 g, 2.86 mmol), H₂O (0.84 mL) and TEA (0.83 mL, 0.60 g,5.93 mmol) in THF (2 mL) at 0° C. (ice/acetone) under nitrogen. Thereaction mixture was allowed to warm to room temperature and stirred fora further 2 hours at which time TLC (EtOAc) revealed reactioncompletion. After removal of the THF by evaporation in vacuo, theresidue was partitioned between H₂O (100 mL) and EtOAc (100 mL). Theaqueous layer was washed with EtOAc (3×50 mL), and the combined organiclayers washed with saturated NH₄CL (100 mL), brine (100 mL), dried(MgSO₄), filtered and concentrated in vacuo to give the crude product asa dark orange oil. Purification by flash chromatography (50%EtOAc/Petroleum Ether) afforded the pure amide 75 as a pale yellow glass(0.93 g, 74%): [α]²¹ _(D)=−51.1° (c=0.08, CHCl₃); ¹H NMR (270 MHz,CDCl₃) (Rotamers) δ7.77 and 7.74 (s×2, 2H_(arom)), 6.81 and 6.76 (s×2,2H_(arom)), 5.09-4.83 (m, 4H, NCH₂C═CH₂), 4.60 (m, 2H, NCHCH₂OTBDMS),4.35-4.31 (m, 4H, OCH₂CH₂CH₂O), 4.08-3.74 (m, 14H, NCHCH₂OTBDMS,NCH₂C═CH₂ and OCH₃), 2.72-2.45 (m, 6H, NCH₂C═CH₂CH₂ and OCH₂CH₂CH₂O),0.91 and 0.79 (s×2, 18H, SiC(CH₃)₃), 0.09, −0.09, and −0.12 (s×3, 12H,Si(CH₃)₂); ¹³C NMR (67.8 MHz, CDCl₃) (Rotamers) δ166.2 (NC═O), 154.7 and154.5 (C_(quat)), 148.4 and 148.2 (C_(quat)), 144.1 and 143.2(C_(quat)), 137.2 (C_(quat)), 128.2 and 127.4 (C_(quat)), 110.1 and108.6 (C—H_(arom)), 109.1 and 108.3 (C—H_(arom)), 107.5 (NCH₂C═CH₂),65.7 and 65.5 (OCH₂CH₂CH₂O), 63.9 and 62.6 (NCHCH₁₂TBDMS), 60.2(NCHCH₂OTBDMS), 58.1 and 56.6 (OCH₃), 52.8 and 50.5 (NCH2C═CH₂), 35.0and 33.9 (NCH₂C═CH₂CH₂), 30.8 and 28.6 (OCH₂CH₂CH₂O), 25.8 and 25.7(SiC(CH₃)₃), 18.2 (SiC(CH₃)₃), −5.5 and −5.6 (Si(CH₃)₂); MS (EI), m/z(relative intensity) 885 (M^(+.), 7), 828 (M—^(t)Bu, 100), 740(M—CH₂OTBDMS, 20), 603 (3), 479 (26), 391 (27), 385 (25), 301 (7), 365(10), 310 (14), 226 (8), 222 (13), 170 (21), 168 (61), 82 (39), 75 (92);IR (NUJOL™) 2923, 2853, 2360, 1647, 1587, 1523 (NO₂), 1461, 1429, 1371,1336 (NO₂), 1277, 1217, 1114, 1061, 1021, 891, 836 772, 739 cm⁻¹.

(2S)-1,1′-[[(Propane-1,3-diyl)dioxy]bis[(2-nitro-5-methoxy-1,4-phenylene)carbonyl]]bis[2-(hydroxymethyl)-4-methylidenepyrrolidine] (76)

A solution of TBAF (3.98 mL of a 1M solution in THF, 3.98 mmol) wasadded to the bis-silyl ether 75 (1.41 g, 1.59 mmol) in THF (35 mL) at 0°C. (ice/acetone). The reaction mixture was allowed to warm to roomtemperature and after a further 30 minutes saturated NH₄Cl (120 mL) wasadded. The aqueous solution was extracted with EtOAc (3×80 mL), washedwith brine (80 mL), dried (MgSO₄), filtered and evaporated in vacuo togive a dark orange oil which was purified by flash chromatography (97%CHCl₃/MeOH) to provide the pure diol 76 as a light orange solid (0.98 g,94%): [α]¹⁹ _(D)=−31.9° (c=0.09, CHCl₃); ¹H NMR (270 MHz, CDCl₃)(Rotamers) δ7.75 and 7.71 (s×2, 2H_(arom)), 6.96 and 6.84 (s×2,2H_(arom)), 5.08, 5.02 and 4.88 (br s×3, 4H, NCH₂C═CH₂), 4.61-4.50 (m,2H, NCHCH₂OH), 4.35-4.33 (m, 4H, OCH₂CH₂CH₂O), 4.02-3.65 (m, 14H,NCHCH₂OH, NCH₂C═CH₂ and OCH,), 2.88-2.43 (m, 6H, NCH₂C═CH₂CH₂ andOCH₂CH₂CH₂O); ¹³C NMR (67.8 MHz, CDCl₃) (Rotamers) δ167.9 and 166.9(NC═O), 154.9 and 154.3 (C_(quat)), 148.4 and 148.2 (C_(quat)), 143.3and 142.6 (C_(quat)), 137.2 and 137.0 (C_(quat)), 127.6 and 127.3(C_(quat)), 109.1 (C—H_(arom)), 108.4 (NCH₂C═CH₂), 108.2 (C—H_(arom)),65.6 and 65.4 (OCH₂CH₂CH₂O), 64.5 and 63.3 (NCHCH₂OH), 60.5 and 60.0(NCHCH₂OH), 56.8 and 56.7 (OCH₃), 52.9 (NCH₂C═CH₂), 35.0 and 34.3(NCH₂C═CH₂CH₂), 29.6 and 28.6 (OCH₂CH₂CH₂O); MS (FAB) (RelativeIntensity) 657 (M^(+.) +1, 10), 639 (M—OH, 2), 612 (1), 544(M—NCH₂CCH₂CH₂CHCH₂OH, 4), 539 (1), 449 (16), 433(9), 404 (8), 236 (32),166 (65), 151 (81), 112 (82), 82 (100); IR (NUJOL®) 3600-3200 (br, OH),2923, 2853, 2360, 1618, 1582, 1522 (NO₂), 1459, 1408, 1375, 1335 (NO₂),1278, 1218, 1061, 908, 810, 757 cm⁻¹.

(2S)-1,1′-[[(Propane-1,3-diyl)dioxy]bis[(2-amino-5-methoxy-1,4-phenylene)carbonyl]]bis[2-(hydroxymethyl)-4-methylidenepyrrolidine](77)

A mixture of the diol 76 (0.98 g, 1.49 mmol) and SnCl₂₀.2H₂O (3.36 g,14.9 mmol) in MeOH (35 mL) was heated at reflux and the progress of thereaction monitored by TLC (90% CHCl₃/MeOH). After 45 minutes, the MeOHwas evaporated in vacuo and the resulting residue was cooled (ice), andtreated carefully with saturated NaHCO₃ (120 mL). The mixture wasdiluted with EtOAc (120 mL), and after 16 hours stirring at roomtemperature the inorganic precipitate was removed by filtration throughcelite. The organic layer was separated, washed with brine (100 mL),dried (MgSO₄), filtered and evaporated in vacuo to give a brown solid.Flash chromatography (95% CHCl₃/MeOH) afforded the pure bis-amine 77 asan orange solid (0.54 g, 61%): [α]¹⁹ _(D)=−31.8° (c=0.30, CHCl₃); ¹H NMR(270 MHz, CDCl₃) δ6.74 (s, 2H_(arom)), 6.32 (s, 2H_(arom)), 5.00 (br s,2H, NCH₂C═CH₂), 4.93 (br s, 2H, NCH₂C═CH₂), 4.54 (br s, 2H, NCHCH₂OH),4.24-4.14 (m, 4H, OCH₂CH₂CH₂O), 3.98-3.50 (m, 14H, NCHCH₂OH, NCH₂C═CH₂and OCH₃), 2.76 (dd, 2H, J=8.61, 15.91 Hz, NCH₂C═CH₂CH₂), 2.46-2.41 (m,2H, NCH₂C═CH₂CH₂), 2.33-2.28 (m, 2H, OCH₂CH₂CH₂O); ¹³C NMR (67.8 MHz,CDCl₃) δ171.0 (NC═O), 151.0 (C_(quat)), 143.5 (C_(quat)), 141.3(C_(quat)), 140.6 (C_(quat)), 112.4 (C—H_(arom)), 111.9 (C_(quat)),107.8 (NCH₂C═CH₂), 102.4 (C—H_(arom)), 65.2 (OCH₂CH₂CH₂O), 65.0(NCHCH₂OH), 59.8 (NCHCH₂OH), 57.1 (OCH₃), 53.3 (NCH₂C═CH₂), 34.4(NCH₂C═CH₂CH₂), 29.0 (OCH₂CH₂CH₂O); MS (FAB) (Relative Intensity) 596(M^(+.), 13), 484 (M—NCH₂CCH₂CH₂CHCH₂OH, 14), 389 (10), 371 (29), 345(5), 224 (8), 206 (44), 166 (100), 149 (24), 112 (39), 96 (34), 81 (28);IR (NUJOL™) 3600-3000 (br, OH), 3349 (NH₂), 2922, 2852, 2363, 1615, 1591(NH₂), 1514, 1464, 1401, 1359, 1263, 1216, 1187, 1169, 1114, 1043, 891,832, 761 cm⁻¹.(2S,4R)&(2S,4S)-1,1′-[[(Propane-1,3-diyl)dioxy]bis[(2-amino-5-methoxy-1,4-phenylene)carbonyl]]bis[2-(hydroxymethyl)-4-methylpyrrolidine](77).

A solution of hydrazine (23 mg, 23 μL, 0.72 mmol) in MeOH (5 mL) wasadded dropwise to a solution of the diol 76 (95 mg, 0.145 mmol) andRaney Ni (20 mg) in MeOH (15 mL) heated at reflux. After 1 hour atreflux TLC (90% CHCl₃/MeOH) revealed some amine formation. The reactionmixture was treated with further Raney Ni (20 mg) and hydrazine (23 mg,23,L, 0.72 mmol) in MeOH (5 mL) and was heated at reflux for anadditional 30 minutes at which point TLC revealed complete reaction. Thereaction mixture was then treated with enough Raney Ni to decompose anyremaining hydrazine and heated at reflux for a further 1.5 hours.Following cooling to room temperature the mixture was filtered through asinter and the resulting filtrate evaporated in vacuo. The resultingresidue was then treated with CH₂Cl₂ (30 mL), dried (MgSO₄), filteredand evaporated in vacuo to provide the bis-amine (77) as a yellow oil(54 mg, 63%): ¹H NMR (270 MHz, CDCl₃) (diastereoisomers) δ6.73 (s,2H_(arom)), 6.32 (s, 2H_(arom)), 4.60-4.30 (m, 2H, NCHCH₂OH), 4.19 (t,4H, J=5.87 Hz, OCH₂CH₂CH₂O), 3.78-3.50 (m, 14H, NCHCH₂OH, NCH₂CHCH₃ andOCH₃), 2.40-1.55 (m, 8H, NCH₂CHCH₃, OCH₂CH₂CH₂O and NCH₂CHCH₃CH₂),1.00-0. 95 (m, 6H, NCH₂CHCH₃); MS (EI), m/z (relative intensity) 600(M^(+.), 16), 459 (46), 345 (16), 206 (13), 186 (17), 180 (31), 166(37), 149 (6), 142 (76), 100 (6), 98 (13), 97 (29), 84 (81), 69 (7), 55(100).

(2S)-1,1′-[[(Propane-1,3-diyl)dioxy]bis[(2-allyloxycarbonylamino-5-methoxy-1,4-phenylene)carbonyl]]bis[2-(hydroxymethyl)-4-methylidenepyrrolidine](78)

Pyridine (0.47 mL, 0.46 g, 5.82 mmol) was added to a stirred solution ofthe bis-amine 77 (0.857 g, 1.44 mmol) in CH₂Cl₂ (30 mL) at 0° C.(ice/acetone). The cool mixture was then treated dropwise with asolution of allyl chloroformate (0.33 mL, 0.38 g, 3.15 mmol) in CH₂Cl₂(10 mL). After 2.5 hours stirring at room temperature, the mixture wasdiluted with CH₂Cl₂ (60 mL), washed with 1N HCl (2×50 mL), H₂O (80 mL),brine (80 mL), dried (MgSO₄), filtered and evaporated in vacuo. Thecrude residue was purified by flash chromatography (70-100%EtOAc/Petroleum Ether) to afford the allyl cartamate compound 78 as aslightly orange glass (0.548 g, 50%): ¹H NMR (270 MHz, CDCl₃) δ8.58 (brs, 2H, NH), 7.56 (s, 2H_(arom)), 6.78 (s, 2H_(arom)), 6.03-5.88 (m, 2H,NCO₂CH₂CH═CH₂), 5.39-5.21 (m, 4H, NCO₂CH₂CH═CH,), 5.00 (br s, 2H,NCH₂C═CH₂), 4.93 (br s, 2H, NCH₂C═CH₂), 4.70-4.57 (m, 4H,NCO₂CH₂CH═CH₂), 4.30-4.25 (m, 4H, OCH₂CH₂CH₂O), 4.17-3.90 (m, 8H,NCHCH₂OH and NCH₂C═CH₂), 3.81-3.54 (m, 8H, NCHCH₂OH and OCH₃), 2.76 (dd,2H, J=8.52, 15.85 Hz, NCH₂C═CH₂CH₂), 2.49-2.44 (m, 2H, NCH₂C═CH₂CH₂),2.36-2.28 (m, 2H, OCH₂CH₂CH₂O); ¹³C NMR (67.8 MHz, CDCl₃) δ170.3(NC═O_(amide)), 153.8 (NC═O_(carbamate)), 150.5 (C_(quat)), 144.8(C_(quat)), 143.1 (C_(quat)) 132.5 (NCO₂CH₂CH═CH₂), 130.7 (C_(quat)),118.1 (NCO₂CH₂CH═CH₂), 116.8 (C_(quat)), 110.9 (C—H_(arom)), 108.1(NCH₂C═CH₂), 106.9 (C—H_(arom)), 65.7 (NCO₂CH₂CH═CH₂), 65.4(OCH₂CH₂CH₂O), 65.1 (NCHCH₂OH), 59.8 (NCHCH₂OH), 56.5 (OCH₃), 53.9(NCH₂C═CH₂), 34.2 (NCH₂C═CH₂CH₂), 29.7 and 29.2 (OCH₂CH₂CH₂O); MS (FAB)(Relative Intensity) 765 (M^(+.) +1, 10), 652 (M—NCH₂CCH₂CH₂CHCH₂OH,32), 594 (4), 539 (2), 481 (51), 441 (31), 290 (3), 249 (13), 232 (38),192 (83), 166 (49), 149 (32), 114 (100).

1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11S,1aS)-10-(allyloxycarbonyl)-11-hydroxy-7-methoxy-2-methylidene-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one](79)

A solution of the bis-alloc compound 78 (150 mg, 0.196 mmol) inCH₂Cl₂/CH₃CN (12 mL, 3:1) was treated with 4 A powdered molecular sieves(0.2 g) and NMO (70 mg, 0.598 mmol). After 15 minutes stirring at roomtemperature, TPAP (7 mg, 19.9 μmol) was added and stirring continued fora further 2 hours at which time TLC (95% CHCl₃/MeOH) indicatedformation, of the fully cyclised product along with the presumedsemi-cyclised product 79, and unreacted starting material 78 present inthe reaction mixture. The mixture was then treated with a furtherquantity of NMO (35 mg, 0.299 mmol) and TPAP (3.5 mg, 9.96 μmol), andallowed to stir for a further 0.5 hours when TLC revealed reactioncompletion. The solvent was evaporated in vacuo and the black residuewas subjected to flash chromatography (98% CHCl₃/MeOH) to provide thepure protected carbinolamine 79 as a white solid (47 mg, 32%): ¹H NMR(270 MHz, CDCl₃) δ7.23 (s, 2H_(arom)), 6.74 (s, 2Ha,), 5.90-5.65 (m, 2H,NCO₂CH₂CH═CH₂), 5.57 (d, 2H, J=8.24 Hz, NCHCHOH), 5.26-5.07 (m, 8H,NCH₂C═CH₂ and NCO₂CH₂CH═CH₂), 4.67-4.10 (m, 14H, NCO₂CH₂CH═CH₂,NCH₂C═CH₂, OCH₂CH₂CH₂O and OH), 3.89 (S, 6H, OCH₃), 3.63 (m, 2H,NCHCHOH), 2.91 (dd, 2H, J=8.79, 15.76 Hz, NCH₂C═CH₂CH₂), 2.68 (d, 2H,J=16.10 Hz, NCH₂C═CH,CH₂), 2.42-2.24 (m, 2H, OCH₂CH₂CH₂O); ¹³C NMR (67.8MHz, CDCl₃) δ166.7 (NC═O_(amide)), 150.1 (C_(quat)), 149.0 (C_(quat)),141.7 (C_(quat)), 131.7 (NCO₂CH₂CH═CH₂), 130.6 (C_(quat)), 128.9(C_(quat)), 128.8 (C_(quat)), 118.3 (NCO₂CH₂CH═CH₂), 114.7 (C—H_(arom)),110.7 (C—H_(arom)), 109.8 (NCH₂C═CH₂), 85.9 (NCHCHOH), 66.9(NCO₂CH₂CH═CH₂), 66.0 (OCH₂CH₂CH₂O), 59.7 (NCHCHOH), 56.1 (OCH₃), 50.7(NCH₂C═CH₂), 35.0 (NCH₂C═CH₂CH₂), 29.7 and 29.1 (OCH₂CH₂CH₂O); MS (FAB)(Relative Intensity) 743 (M^(+.) −17, 16), 725 (17), 632 (13), 574 (8),548 (13), 490 (10), 481 (9), 441 (7), 425 (6), 257 (12), 232 (20), 192(46), 166 (52), 149 (100), 91 (59); IR (NUJOL®) 3234 (br, OH), 2923,2853, 2361, 1707, 1604, 1515, 1464, 1410, 1377, 1302, 1267, 1205, 1163,1120, 1045, 999, 955, 768, 722 cm⁻¹.

1,1′-[[(Propane-1,3-diyl)dioxy]bis[(11aS)-7-methoxy-2-methylidene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one](80, SJG-136)

A catalytic amount of tetrakis(triphenylphosphine)palladium (11 mg, 9.52μmol) was added to a stirred solution of the bis-alloc-carbinolamine 79(139 mg, 0.183 mmol), triphenylphosphine (4.8 mg, 18.3 μmol) andpyrrolidine (27 mg, 0.380 mmol) in CH₂Cl₂/CH₃CN (13 mL, 10:3) at 0° C.(ice/acetone) under a nitrogen atmosphere. The reaction mixture wasallowed to warm to room temperature and the progress monitored by TLC(95% CHCl₃/MeOH). After 2 hours 15 minutes TLC revealed the reaction wascomplete, proceeding via the presumed half-imine product 261, to give aTLC spot which fluoresced brightly under UV. The solvent was evaporatedin vacuo and the resulting residue subjected to flash chromatography(98% CHCl₃/MeOH) to give the bis-imine target molecule 80 (SJG-136) as apale orange glass (78 mg, 77%) which was repeatedly evaporated in vacuowith CHCl₃ to provide the imine form: [α]²¹ _(D)=+357.7° (c=0.07,CHCl₃); Reverse Phase HPLC (C, stationary phase, 65% MeOH/H₂O mobilephase, 254 nm), Retention time 6.27 minutes, % Peak area =97.5%; ¹H NMR(270 MHz, CDCl₃) (imine form) δ7.68 (d, 2H, J=4.4 Hz, HC═N), 7.49 (S,2H_(arom)), 6.85 (s, 2H_(arom)), 5.20 (s, 2H, NCH₂C═CH₂), 5.17 (s, 2H,NCH₂C—CH₂), 4.46-4.19 (m, 4H, OCH₂CH₂CH₂O), 3.92 (S, 6H, OCH₃),3.89-3.68 (m, 6H, NCH₂C═CH₂ and NCHHC═N), 3.12 (dd, 2H, J=8.61, 16.21Hz, NCH₂C═CH₂CH₂), 2.68 (d, 2H, J=16.30 Hz, NCH₂C═CH₂CH₂), 2.45-2.38 (m,2H, OCH₂CH₂CH₂O); ¹³C NMR (67.8 MHz, CDCl₃) (imine form) δ164.7 (NC═O),162.6 (HC═N), 150.7 (C_(quat)), 147.9 (C_(quat)), 141.5 (C_(quat)),140.6 (C_(quat)), 119.8 (C_(quat)), 111.5 (C—H_(arom)), 110.7(C—H_(arom)), 109.4 (NCH₂C═CH₂), 65.4 (OCH₂CH₂CH₂O), 56.1 (OCH₃), 53.8(NCHHC═N), 51.4 (NCH₂C═CH₂), 35.4 (NCH₂C═CH₂CH₂), 28.8 (OCH₂CH₂CH₂O); MS(FAB) (Relative Intensity) (imine form) 773 (M^(+.) +1+(Thioglyceroladduct X 2), 3), 665 (M^(+.) +1+Thioglycerol adduct, 7), 557 (M^(+.) +1,9), 464 (3), 279 (12), 257 (5), 201 (5), 185 (43), 166 (6), 149 (12), 93(100); IR (NUJOL®) 3600-3100 (br, OH of carbinolamine form), 2923, 2849,1599, 1511, 1458, 1435, 1391, 1277, 1228, 1054, 1011, 870, 804, 761, 739cm⁻¹.

Alternative Synthesis of UP2001, SJG-136 (80) (See FIG. 11)

UP2001 was also prepared by an alternative synthesis based the bis-ketone 52 (See Example 11(f)).

1,1′-[[(Propane-1,3-diyl)dioxy]bis[2-amino-N-allyloxycarbonyl-5-methoxy-1,4-phenylene)carbonyl]]-bis[(2S)-2-t-butyldimethylsilyloxymethyl-4-methylidene-2,3-dihydropyrrole](206)

A solution of potassium-t-butoxide in dry THF (0.5 M, 4.00 mL, 2.00mmol) was added to as suspension of methyltriphenylphosphonium bromide(0.716 g, 2.00 mmol) in dry THF (2.00 mL). The resulting yellow ylidesuspension was allowed to stir at 0° C. for 2 hours before the additionof a solution of the bis-ketone 52 (0.50 g, 0.50 mmol) in THF (10 mL) at10° C. The reaction mixture was allowed to warm to room temperature andstirring was continued for a further hour. The reaction mixture waspartitioned between ethyl acetate (15 mL) and water (15 mL) and theorganic layer was washed the sat. sodium chloride (20 mL) and dried overmagnesium sulphate. Removal of excess solvent gave a brown oil that wassubjected to flash column chromatography (50% ethyl acetate, 50% 40-60°petroleum ether) to afford the product as a yellow glass 206 (250 mg,51%). [α]^(23.4) _(D)=−32° (c 0.265, CHCl₃). ¹H NMR (CDCl₃): δ0.00 (s,12H), 0.88 (s, 18H), 2.37-2.40 (m, 2H), 2.69-2.75 (m, 4H), 3.80-4.62 (m,20H), 4.61-4.63 (m, 4H), 4.98 (bs, 4H), 5.30-5.38 (m, 4H), 5.94-6.00 (m,2H), 6.81 (S, 2H), 7.84 (s, 2H), 8.80 (bs, 2H).

1,1′-[[(Propane-1,3-diyl)dioxy]bis[2-amino-N-allyloxycarbonyl-5-methoxy-1,4-phenylene)carbonyl]]-bis[(2S)-2-hydroxymethyl-4-methylidene-2,3-dihydropyrrole](78)

An aliquot of hydrogen fluoride/pyridine complex (0.8 mL, 70% HF, 30%pyridine) was added to a solution of the bis-silyl ether 206 (285 mg,0.287 mmol) in THF (10 mL) at 0° C. under a nitrogen atmosphere.Stirring was continued at 0° C. for 30 minutes and the reaction mixturewas then allowed to rise to room temperature over a 1 hour period. Thereaction mixture was neutralised with sodium bicarbonate and extractedwith dichloromethane (3×30 mL). The combined organic phase was washedwith brine and dried over magnesium sulphate. Removal of excess solventunder reduced pressure afforded the product 78 as a yellow gum (218 mg).

1,1′[[(Propane-1,3-diyl)dioxy]bis(11S,11aS)-10-(allyloxycarbonyl)-11-hydroxy-7-methoxy-2-methylidene-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4-benzodiazepin-5-one](79)

A solution of dimethyl sulphoxide (0.55 mL, 7.75 mmol) in drydichloromethane (10 mL) was added dropwise, over a 15 minute period, toa stirred solution of oxalyl chloride (0.32 mL, 3.67 mmol) indichloromethane (10 mL) at −45° C. under a nitrogen atmosphere. Thereaction mixture was allowed to stir for 35 minutes at −45° C. followedby addition of the diol 78 (1.01 g, 1.32 mmol) in dichloromethane (10mL), at the same temperature, over 15 minutes. After a further 45minutes a solution of triethylamine (1.50 mL, 10.76 mmol) indichloromethane (10 mL) was added over a period of 15 minutes. Thereaction mixture was allowed to stir at −45° C. for 30 minutes beforebeing allowed to warm to room temperature over 45 minutes. The reactionmixture was diluted with water and the phases were allowed to separate.The organic phase was washed with 1M HCl (3×50 mL), sat. sodium chloride(50 mL) and dried over magnesium sulphate. Removal of excess solventyielded the crude product, which was purified by flash columnchromatography (1.5% methanol, 98.5% chloroform) to afford the product79 (0.785 g, 77%).

1,1′[[(propane-1,3-diyl)dioxy]bis[(11aS)-7-methoxy-2-methylidene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazein-5-one](80, SJG-136)

A catalytic amount of tetrakis(triphenylphosphine)palladium (21 mg,0.018 mmol) was added to a stirred solution of thebis-alloc-carbinolamine 79 (250 mg, 0.33 mmol), triphenylphosphine (10mg, 0.033 mmol) and pyrrolidine (0.05 mL, 0.66 mmol) in dry CH₂Cl₂ (30mL) at 0° C. (ice/acetone) under a nitrogen. atmosphere. The reactionmixture was allowed to stir for 2 hours before warming to roomtemperature over 1 hour. The solvent was evaporated under reducedpressure and the resulting residue subjected to flash chromatography(98% CHCl₃/MeOH) to give the bis-imine target molecule 80 (SJG-136).

Example 2(e)

Synthesis of1,1′[[(pentane-1,5-diyl)dioxy]bis[(11aS)-7-methoxy-2-methylidene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one](218) (See FIGS. 12 a/b)

Preparation of Nitro Dimer Core

1′,5′-Bis[2-methoxy-4-(methoxycarbonyl)phenoxy]pentane (208)

Neat diethyl azidodicarboxylate (19.02 mL, 21.04 g, 120.8 mmol) wasadded dropwise over 30 minutes to a stirred solution of methyl vanillate(206) (20 g, 109.8 mmol) and triphenylphosphine (43.2 g, 164.7 mmol) inanhydrous THF (400 mL) and the reaction mixture was allowed to stir at0° C. for 1 h. The cold reaction mixture was treated dropwise over 20minutes with a solution of 1,5-pentanediol (207) (3.83 mL, 4.03 g, 53.0mmol) in THF (4 mL). The reaction mixture was allowed to stir overnightat room temperature and the precipitated product (208) was collected byvacuum filtration. Dilution of the filtrate with methanol precipitatedfurther product (208). The combined precipitate (12.3 g, 52% based onpentanediol) was used in the next step without further purification: ¹HNMR (270 MHz, CDCl₃) δ7.65 (dd, 2H, J=2.01, 8.42 Hz), 7.54 (d, 2H,J=2.01 Hz), 6.87 (d, 2H, J=8.42 Hz), 4.10 (t, 4H, J=6.59 Hz), 3.90 (s,6H), 3.89 (s, 6H), 2.10-1.90 (m, 4H), 1.85-1.26 (m, 2H).

1′,5′-Bis[2-methoxy-4-(methoxycarbonyl)-5-nitrophenoxy]pentane (209)

Solid copper (II) nitrate trihydrate (16.79 g, 69.5 mmol) was addedslowly to a stirred solution of the bis-ester (208) (12 g, 27.8 mmol) inacetic anhydride (73 mL) at 0° C. The reaction mixture was allowed tostir for 1 hour at 0° C., the ice bath was removed and the reactionmixture was allowed to warm to room temperature a mild exotherm, c. 40°C., accompanied by the evolution of NO₂occurred at this stage. After theexotherm had subsided stirring at room temperature was continued for 2hours. The reaction mixture was poured into ice water and the aqueoussuspension allowed to stir for 1 h. The resulting yellow precipitate wascollected by vacuum filtration and dried in air to afford the desiredbis nitro compound (209) (14.23 g, 98%): ¹H NMR (400 MHz, CDCl₃+DMSO)δ7.45 (s, 2H), 7.09 (s, 2H), 4.14 (t, 4H, J=6.31 Hz), 3.97 (s, 6H), 3.90(s, 6H), 2.20-1.94 (m, 4H), 1.75-1.70 (m, 2H).

1′,5′-Bis(4-carboxy-2-methoxy-5-nitrophenoxy)pentane (210)

A suspension of the ester 209 (9.0 g, 17.2 mmol) in aqueous sodiumhydroxide (1 M, 180 mL) and THF (180 mL) was allowed to stir until ahomogenous solution was obtained (2 days). THF was evaporated underreduced pressure and the resulting aqueous suspension was filtered toremove any unreacted starting material. The filtrate was adjusted to pH1, the precipitated product was collected by filtration and air dried toafford the desired bis-acid (210) (8.88 g). A higher than theoreticalyield was obtained due to the inclusion of the sodium salt of acid. Thesalt may be removed by dissolving the bulk of the material in THF andremoving the insoluble material by filtration: ¹H NMR (400 MHz, CDCl₃)δ7.39 (s, 2H), 7.16 (s, 2H), 4.12 (t, 4H, J=6.59 Hz), 3.95 (s, 6H),2.00-1.85 (m, 4H), 1.75-1.67 (m, 2H).

Assembling the Bis Ketone Intermediate

1,1′-[[(Pentane-1,5-diyl)dioxy]bis[2-nitro-5-methoxy-1,4-phenylene)carbonyl]]-bis[(2S,4R)-2-t-butyldimethylsilyloxymethyl-4-hydroxypyrrolidine](211)

A catalytic amount of DMF (5 drops) was added to as stirred suspensionof the acid 210 (5.39 g, 10.9 mmol) and oxalyl chloride (3.47 g, 2.38mL, 27.3 mmol) in anhydrous THF (50 mL). Initial effervescence wasobserved followed by the formation of a homogenous solution, howeverafter stirring overnight a suspension of the newly formed acid chloridewas formed. Excess THF and oxalyl chloride was removed by rotaryevaporation under reduced pressure and the acid chloride was resuspendedin fresh THF (49 mL). The acid chloride solution was added dropwise to asolution of the (2S,4R)-2-t-butyldimethylsilyloxymethyl-4-hydroxypyrrolidine (2) (6.3 g,27.3 mmol), triethylamine (4.42 g, 6.09 mL, 43.7 mmol) and water (1.47mL) in THF (33 mL) at 0° C. under a nitrogen atmosphere. The reactionmixture was allowed to warm to room temperature and stirring wascontinued for 3 h. Excess THF was removed by rotary evaporation underreduced pressure and the resulting residue was partitioned between water(300 mL) and ethyl acetate (300 mL). The layers were allowed to separateand the aqueous layer was extracted with ethyl acetate (3×150 mL). Thecombined organic layers were then washed with ammonium chloride (150mL), sat. sodium bicarbonate (150 mL), brine (150 mL) and dried overmagnesium sulphate. Filtration followed by rotary evaporation underreduced pressure afforded the crude product as a dark oil. The crudeproduct was subjected to flash column chromatography (3% methanol, 97%chloroform) and removal of excess eluent isolated (211) (3.70 g, 37%yield): ¹H NMR (270 MHz, CDCl₃) δ7.65 (s, 2H), 6.77 (s, 2H), 4.52 (bs,2H), 4.40 (bs, 2H), 4.17-4.10 (m, 6H), 3.92 (s, 6H), 3.77 (d, 2H,J=10.26 Hz), 3.32 (td, 2H, J=4.40, 11.35 Hz), 3.08 (d, 2H, J=11.35 Hz),2.37-2.27 (m, 2H), 2.10-2.00 (m, 6H), 1.75-1.60 (m, 2H), 0.91 (s, 18H),0.10 (s, 12H).

1,1′-[[(Pentane-1,5-diyl)dioxy]bis[2-amino-5-methoxy-1,4-phenylene)carbonyl]]-bis[(2S,4R)-2-t-butyldimethylsilyloxymethyl-4-hydroxypyrrolidine](212)

A methanolic solution of hydrazine hydrate (1.25 mL, 1.29 g, 40.2 mmolof hydrazine, 20 mL of methanol) was added dropwise to a solution of thebis-nitro compound 211 (3.6 g, 3.91 mmol) in methanol (68 mL) gentlyrefluxing over Raney nickel (510 mg of a thick slurry). After 5 minutesat reflux TLC (10% MeOH, 90% chloroform) revealed the incompleteconsumption of starting material. The reaction mixture was treated withadditional Raney nickel (c 510 mg) and hydrazine (1.25 mL) in methanol(20 mL) resulting in complete consumption of starting material. ExcessRaney nickel was added to the reaction mixture to decompose unreactedhydrazine hydrate and the reaction mixture was then allowed to cool. Thereaction mixture was filtered through celite to remove excess Raneynickel and the filter pad washed with additional methanol (Caution!Raney nickel is pyrophoric, do not allow filter pad to dry, use conc.HCl to destroy nickel). The combined filtrate was evaporated by rotaryevaporation under reduced pressure and the residue re-dissolved indichloromethane. The dichloromethane solution was dried over magnesiumsulphate (to remove water associated with the hydrazine), filtered andevaporated to afford the product (212) as a foam (3.37 g, 91%): ¹H NMR(270 MHz, CDCl₃) δ6.69 (s, 2H), 6.24 (s, 2H), 4.40-3.40 (m, 28H),2.40-1.60 (m, 10H), 0.88 (s, 18H), 0.03 (s, 12H).

1,1′-[[(Pentane-1,5-diyl)dioxy]bis[2-amino-N-allyloxycarbonyl-5-methoxy-1,4-phenylene)carbonyl]]-bis[(2S,4R)-2-t-butyldimethylsilyloxymethyl-4-hydroxypyrrolidine](213)

A solution of allyl chloroformate (0.806 mL, 0.916 g, 7.6 mmol) in drydichloromethane (63 mL) was added, dropwise, to a solution of thebis-amine 212 (3.27 g, 3.8 mmol) and pyridine (1.26 g, 1.29 mL, 15.9mmol) in dichloromethane (128 mL) at 0° C. under a nitrogen atmosphere.The reaction mixture was allowed to warm to room temperature and to stirfor 16 h. At which time TLC (10% MeOH, 90% Chloroform) revealed reactionto be complete. The reaction mixture was diluted with dichloromethane(40 mL) and washed with sat. copper II sulphate (2×140 mL), water (120mL) and sat. sodium chloride (120 mL). The organic phase was dried overmagnesium sulphate, filtered and evaporated under reduced pressure toafford 213 as a foam (3.60 g, 92%): ¹H NMR (270 MHz, CDCl₃) δ8.87 (bs,2H), 7.66 (s, 2H), 6.77 (s, 2H), 6.05-5.80 (m, 2H), 5.40-5.15 (m, 4H),4.70-4.50 (m, 6H), 4.38 (bs, 2H), 4.20-4.00 (m, 4H), 3.78 (s, 6H),3.70-3.40 (m, 8H), 2.40-2.20 (m, 2H), 2.10-1.80 (m, 6H), 1.75-1.55 (m,2H), 0.89 (s, 18H), 0.04 (s, 12H).

1,1′-[[(Pentane-1,5-diyl)dioxy]bis[2-amino-N-allyloxycarbonyl-5-methoxy-1,4-phenylene)carbonyl]]-bis[(2s)-2-t-butyldimethylsilyloxymethyl-4-oxo-pyrrolidine](214)

A solution of dimethyl sulphoxide (1.47 mL, 1.62 g, 20.7 mmol) in drydichloromethane (32 mL) was added dropwise over 45 minutes to a stirredsolution of oxalyl chloride (5.18 mL of a 2 M solution indichloromethane, 10.35 mmol) at −60° C. under a nitrogen atmosphere.After stirring at −50° C. for 30 minutes, a solution of the bis-alcohol213 (3.55 g, 3.45 mmol) in dichloromethane (53 mL) was added dropwiseover a period of 50 minutes. The reaction mixture was allowed to stir at−60° C. for 30 minutes prior to the dropwise addition of a solution oftriethylamine (4.75 g, 6.54 mL, 46.9 mmol) in dichloromethane (27 mL).Stirring was continued at −60° C. for 45 minutes and then allowed towarm to 0° C. The reaction mixture was diluted with dichloromethane (20mL), washed with cold 1 M HCl (2×100 mL), sat. sodium chloride (100 mL)and dried over magnesium sulphate. Removal of excess solvent affordedthe crude bis-ketone which was purified by flash column chromatography(50% ethyl acetate, 50% 40-60° petroleum ether) to yield the purebis-ketone (214) as a pale yellow foam (2.54 g, 72%): ¹H NMR (270 MHz,CDCl₃) δ8.69 (bs, 2H), 7.78 (s, 2H), 6.75 (s, 2H), 6.05-5.80 (m, 2H),5.40-5.20 (m, 4H), 4.65-4.60 (m, 4H), 4.20-3.60 (m, 20H), 2.74 (dd, 2H,J=9.25, 18.1 Hz), 2.51 (d, 2H, J=17.4 Hz), 2.00-1.90 (m, 4H), 1.75-1.65(m, 2H), 0.87 (s, 18H), 0.05 (s, 12H).

Elaboration of Bis Ketone and Preparation of the Target Molecule

1,1′-[[(Pentane-1,5-diyl)dioxy]bis[2-amino-N-allyloxycarbonyl-5-methoxy-1,4-phenylene)carbonyl]]-bis[(2aS)-2-t-butyldimethylsilyloxymethyl-4-methylidene-2,3-dihydropyrrole](215)

A solution of potassium-t-butoxide in dry THF (0.5 M, 25.2 mL, 12.6mmol) was added dropwise to a suspension of methyltriphenylphosphoniumbromide (4.50 g, 12.6 mmol) in dry THF (15 mL). The resulting yellowylide suspension was allowed to stir at 0° C. for 2 hours before theaddition of a solution of the bis-ketone 214 (2.48 g, 2.42 mmol) in THF(10 mL) at 10° C. The reaction mixture was allowed to warm to roomtemperature and stirring was continued for a further hour. The reactionmixture was partitioned between ethyl acetate (100 mL) and water (100mL) and the organic layer was washed with sat. sodium chloride (200 mL)and dried over magnesium sulphate. Removal of excess solvent gave abrown oil that was subjected to flash column chromatography (50% ethylacetate, 50% 40-60° petroleum ether) to afford the product (215) as ayellow glass (865 mg, 35%): ¹H NMR (400 MHz, CDCl₃) δ8.90 (bs, 2H), 7.83(s, 2H), 6.82 (s, 2H), 6.05-5.90 (m, 2H), 5.40-5.20 (m, 4H), 4.99 (bs,2H), 4.91 (bs, 2H), 4.65-4.60 (m, 4H), 4.20-3.60 (m, 20H), 2.70 (bs,4H), 2.00-1.90 (m, 4H), 1.75-1.63 (m, 2H), 0.88 (s, 18H), 0.03 (s, 12H).

1,1′-[[(Pentane-1,5-diyl)dioxy]bis[2-amino-N-allyloxycarbonyl-5-methoxy-1,4-phenylene)carbonyl]]-bis[(2S)-2-hydroxymethyl-4-methylidene-2,3-dihydropyrrole](216)

A solution of TBAF (3.02 mL of a 1 M solution in THF, 3.02 mmol) wasadded to the bis-silyl ether (215) (1.23 g, 1.21 mmol) in THF (30 mL) at0° C. (ice/acetone). The reaction mixture was allowed to warm to roomtemperature and to stir overnight, the following day, TLC (50:50EtOAc/Pet-Ether 40 °-60°) revealed the complete disappearance ofstarting material. Saturated NH₄Cl (150 mL) was added and the reactionmixture extracted with EtOAc (3×60 mL), washed with sat. sodium chloride(150 mL), dried (MgSO₄), filtered and evaporated in vacuo to give ayellow oil. Purification by flash chromatography (97% CHCl₃/3% MeOH)provided the pure alcohol (216) (916 mg, 96%): ¹H NMR (400 MHz, CDCl₃)δ8.61 (bs, 2H), 7.58 (s, 2H), 6.79 (s, 2H), 6.05-5.90 (m, 2H), 5.40-5.20(m, 4H), 5.01 (bs, 2H), 4.93 (bs, 2H), 4.65-4.60 (m, 4H), 4.20-3.60 (m,20H), 2.76 (dd, 2H, , J=8.42, 15.74 Hz), 2.47 (d, 2H, J=15.93 Hz),2.00-1.90 (m, 4H), 1.80-1.63 (m, 2H).

1,1′[[(Pentane-1,5-diyl)dioxy]bis(11S,11aS)-10-(allyloxycarbonyl)-11-hydroxy-7-methoxy-2-methylidene-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4-benzodiazepin-5-one](217)

A solution of dimethyl sulphoxide (0.57 mL, 0.63 g, 8.07 mmol) in drydichloromethane (17 mL) was added dropwise, over a 40 minute period, toa stirred solution of oxalyl chloride (2.02 mL, of a 2 M solution, 4.04mmol) at −45° C. under a nitrogen atmosphere. The reaction mixture wasallowed to stir for 40 minutes at −45° C. followed by addition of thediol 216 (0.89 g, 1.12 mmol) in dichloromethane (17 mL), at the sametemperature, over 15 minutes. After a further 60 minutes a solution oftriethylamine (1.31 mL, 9.42 mmol) in dichloromethane (9 mL) was addedover a period of 40 minutes. The reaction mixture was allowed to stir at−45° C. for 40 minutes before being allowed to warm to room temperatureover 45 minutes. The reaction mixture was diluted with water and thephases were allowed to separate. The organic phase was washed with 1 MHCl (2×40 mL), water (40 mL), sat. sodium chloride (40 mL) and driedover magnesium sulphate. Removal of excess solvent yielded the crudeproduct, which was purified by flash column chromatography (1% methanol,99% chloroform) to afford the product 217 (0.175 g, 20%): ¹H NMR (400MHz, CDCl₃) δ7.22 (s, 2H), 6.65 (s, 2H), 5.82-5.70 (m, 2H), 5.58 (d, 2H,J=9.70 Hz), 5.25-5.00 (m, 8H), 5.75-4.35 (m, 4H), 4.30 (d, 2H, J=16.10Hz), 4.15 (d, 2H, J=17.03 Hz), 4.01 (t, 4H, J=6.32 Hz), 3.90 (s, 6H),3.64 (t, 2H, J=8.70 Hz), 3.00-2.85 (m, 2H), 2.71 (d, 2H, J=16.29 Hz),2.00-1.85 (m, 4H), 1.70-1.60 (m, 2H).

1,1′[[(pentane-1,5-diyl)dioxy]bis[(11aS)-7-methoxy-2-methylidene-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one](218)

A catalytic amount of tetrakis(triphenylphosphine)palladium (13 mg, 11.2mmol) was added to a stirred solution of the bis-alloc-carbinolamine(217) (170 mg, 0.22 mmol), triphenylphosphine (5.7 mg, 21.6 mmol) andpyrrolidine (31 mg, 37.3 mL 0.45 mmol) in DCM (13 mL) at 0° C.(ice/acetone) under a nitrogen. atmosphere. The reaction mixture wasallowed to warm to room temperature and the progress of reactionmonitored by TLC (95% CHCl₃/MeOH). After 2 hours TLC revealed thereaction was complete to give a spot, which fluoresced brightly under UVlight. The solvent was evaporated under reduced pressure and theresulting residue subjected to flash chromatography (99% to 98CHCl₃/MeOH) to give the bis-imine target molecule 218 as a pale yellowglass (84.5 mg, 75%) which was repeatedly evaporated in vacuo with CHCl₃to provide the imine form: ¹H NMR (400 MHz, CDCl₃) δ7.68 (d, 2H, J=4.39Hz), 7.49 (s, 2H), 6.80 (s, 2H), 5.19 (bs, 2H), 5.16 (bs, 2H), 4.28 (bs,4H), 4.15-4.00 (m, 4H), 3.92 (s, 6H), 3.90-3.80 (m, 2H), 3.12 (dd, 2H, ,J=8.97, 15.93 Hz), 2.95 (d, 2H, J=15.93 Hz), 2.00-1.85 (m, 4H),1.72-1.67 (m, 2H).

Example 2(f)

Synthesis of PBD with Ketone on C-Ring (172, UP-2067) (See FIG. 13)

(2S)(4R)-N-[4-benzyloxy-5-methoxy-2-(2′,2′,2′-trichloroethoxy)carbonyl]-2-(tert-butyldimethylsilyloxymethyl)-4-hydroxypyrrolidine(168)

A solution of 2,2,2-trichloroethylchloroformate (8.74 g, 5.68 mL, 41.2mmol) in dichloromethane (50 mL) was added to a solution of 4 (18.2 g,37.5 mmol) and pyridine (5.92 g, 6.1 mL, 75.0 mmol) in drydichloromethane (200 mL) at 0° C. under a nitrogen atmosphere. Thereaction mixture was allowed to stir overnight at room temperature andwas then washed with saturated copper sulphate solution (100 mL), water(100 mL) and brine (100 mL). The organic phase was dried over magnesiumsulphate, filtered and excess solvent removed by rotary evaporation toafford the product 168 (22.01 g, 33.2 mmol, 89%) which was used in thesubsequent reaction without further purification. ¹H NMR (270 MHz,CDCl₃) δ9.31 (bs, 1H); 7.48 (s, 1H); 7.45-7.28 (m, 5H); 6.82 (s, 1H);5.17 (bs, 2H); 4.89 (d, J=11.9 Hz, 1H); 4.70 (d, J=11.9 Hz, 1H); 4.56(bs, 1H); 4.40 (bs, 1H); 4.20-4.00 (m, 1H); 3.95-3.40 (m, 7H); 2.40-2.00(m, 2H); 0.09 (s, 9H); 0.04 (s, 6H). ¹³C NMR (67.8 MHz, CDCl₃) δ169.2,152.1, 150.2, 136.1, 128.6, 128.1, 127.7, 111.6, 106.2, 95.2, 74.4,70.7, 70.5, 62.1, 57.2, 56.4, 35.4, 25.8, 18.1, −5.46.

(2S)-N-[4-benzyloxy-5-methoxy-2-(2′,2′,2′-trichloroethoxy)carbonylamino]-2-(tert-butyldimethylsilyloxymethyl)-4-oxopyrrolidine (169)

A solution of DMSO (7.80 g, 99.8 mmol) in dry dichloromethane (18 mL)was added dropwise, over 30 minutes, to a solution of oxalyl chloride(6.34 g, 49.9 mmol) in dry dichloromethane (25 mL) at −45° C. under anitrogen atmosphere and the reaction mixture allowed to stir for afurther 15 minutes. A solution of the substrate 168 (22.01 g, 33.3 mmol)in dichloromethane (50 mL) was added dropwise over 40 minutes to thereaction mixture, which was then allowed to stir for 45 minutes at −45°C. Finally, neat triethylamine (23.52 g, 232.9 mmol) was added dropwiseover 30 minutes and the reaction mixture allowed to stir at −45° C. for15 minutes. The reaction mixture was allowed to warm to roomtemperature, diluted with water (150 mL) and the organic phase washedwith dilute HCl (1N, 100 mL), water (100 mL) and brine (100 mL). Theorganic phase was dried over magnesium sulphate, filtered andconcentrated in vacuo to afford the crude product which was subjected tocolumn chromatography (ethyl acetate/40-60 petroleum ether, 50:50).Removal of excess eluent afforded the product (20.15 g, 92% yield). ¹HNMR (270 MHz, CDCl₃) δ7.88 (bs, 1H); 7.49-7.28 (m, 5H); 6.80 (s, 1H);5.22 (d, J=12.1 Hz, 1H); 5.17 (d, J=12.1 Hz, 1H); 4.80 (bs, 2H);4.10-3.60 (m, 8H); 2.75 (dd, J 18.0, 9.5 Hz, 1H); 2.52 (d, J=18.0 Hz,1H); 0.87 (s, 9H); 0.06 (s, 3H); 0.05 (s, 3H). ¹³C NMR (67.8 MHz)δ208.7, 168.8, 151.8, 150.6, 144.7, 136.0, 128.5, 128.1, 127.7, 110.9,106.4, 95.2, 74.4, 70.7, 66.0, 56.8, 56.4, 39.4, 25.8, 18.0, −5.7.

(2S)-N-[4-benzyloxy-5-methoxy-2-(2′,2′,2′-trichloroethoxy)carbonylamino]-2-(hydroxymethyl)-4-oxopyrrolidine (170)

Glacial acetic acid (60 mL) and water (20 mL) were added to a solutionof ketone 169 (9.44 g, 14.3 mmol) in THF (20 mL) and the reactionmixture allowed to stir for 3 hr. (reaction complete by TLC). Thereaction mixture was diluted with dichloromethane (200 mL) andneutralized dropwise with sat. sodium bicarbonate (1.5 L) in a 5 L flask(effervescence!). The phases were allowed to separate and the aqueouslayer extracted with dichloromethane (2×100 mL). The combined organiclayers were washed with brine and dried over magnesium sulphate. Removalof excess solvent afforded the crude product which was subjected tocolumn chromatography on silica (ethyl acetate/40-60 petroleum ether,50:50) to give the pure product (6.44 g, 83%). ¹H NMR (270 MHz, CDCl₃)δ8.77 (bs, 1H); 7.57 (s, 1H); 7.46-7.28 (m, 5H); 6.83 (s, 1H), 5.13 (s,2H); 4.85-4.70 (m, 3H); 4.07-3.60 (m, 7H); 2.77 (dd, J=18.5, 9.5 Hz,1H); 2.54 (d, J=18.5 Hz, 1H). ¹³C NMR (67.8 MHz, CDCl₃) δ209.0, 169.4,152.3, 150.6, 145.5, 136.0, 130.0, 128.6, 128.3, 127.6, 110.9, 107.4,95.2, 74.5, 70.8, 64.4, 60.4, 56.6, 55.9, 39.5.

(11s,11aS)-4-benzyloxy-11-hydroxy-5-methoxy-4-oxo-10-(2′,2′,2′-trichloroethoxy)carbonyl-amino1,10,11,11a-tetrahydro-5H-pyrrolo-[2,1-c][1,4]benzodiazepin-5-one (171)

A solution of DMSO (4.45 g, 4.04 mL, 56.9 mmol) in dry dichloromethane(25 mL) was added dropwise, over 5 minutes, to a solution of oxalylchloride (3.58 g, 49.9 mmol) in dry dichloromethane (14 mL) at −60° C.under a nitrogen atmosphere and the reaction mixture allowed to stir fora further 15 minutes. A solution of the substrate 170 (10.93 g, 20.0mmol) in dichloromethane (25 mL) was added dropwise over 30 minutes tothe reaction mixture, which was then allowed to stir for 30 minutes at−60° C. Finally, neat triethylamine (11.15 g, 232.9 mmol) was addeddropwise over 30 minutes and the reaction mixture allowed to stir at−60° C. for 15 minutes. The reaction mixture was allowed to warm to roomtemperature, diluted with water (150 mL) and the organic phase washedwith dilute HCl (1N, 100 mL), water (100 mL) and brine (100 mL). Theorganic phase was dried over magnesium sulphate, filtered andconcentrated in vacuo to afford the crude product which was subjected tocolumn chromatography (ethyl acetate/40-60 petroleum ether, 50:50).Removal of excess eluent afforded the product 171 (9.66 g, 89% yield).¹H NMR (270 MHz, CDCl₃) δ7.45-7.33 (m, 5H); 7.27 (s, 1H); 6.95 (s, 1H);5.76 (d, J=9.9 Hz, 1H); 5.52-5.00 (m, 3H), 4.33 (d, J=6.8 Hz, 1H); 4.30(d, J=19.2 Hz, 1H); 4.00-3.70 (m, 5H); 2.98 (dd, J=20.0, 10.4 Hz, 1H);2.94 (d, J=20.0 Hz, 1H). ¹³C NMR (67.8 MHz) δ207.7, 167.5, 154.5, 152.6,150.8, 149.6, 135.8, 128.9-127.3, 124.0, 114.5, 110.8, 95.0, 86.6, 75.0,71.1, 56.8, 56.2, 52.6, 40.2.

(11aS)-4-benzyloxy-5-methoxy-4-oxo-1,10,11,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(172)

Cadmium/lead couple (1.15 g) was added to a solution of cyclized ketone(1 g, 1.84 mmol) in THF (5 mL) and aqueous ammonium acetate (1N, 15 mL).The reaction mixture was allowed to stir for 90 minutes and thenfiltered through celite. The celite pad was washed with ethyl acetate(2×25 mL) and the organic layer separated. The organic layer was washedwith brine (50 mL) and dried over magnesium sulphate. Removal of excesssolvent followed by column chromatography afforded thepyrrolobenzodiazepine 172 (0.324 g, 0.93 mmol). ¹H NMR (270 MHz, CDCl₃)δ7.75 (d, J=4.4 Hz, 1H); 7.51 (s, 1H); 7.46-7.27 (m, 5H); 5.23 (d,J=12.3 Hz, 1H); 5.17 (d, J=12.3 Hz, 1H), 4.24-4.40 (m, 3H), 3.96 (s,3H), 3.12 (dd, J=19.6, 8.8 Hz, 1H); 2.99 (dd, J=5.0 Hz, 1H). ¹³C NMR(67.8 MHz) δ206.7, 165.5, 161.4, 151.1, 148.5, 140.5, 136.0,128.7-127.1, 118.9, 111.7, 111.3, 70.9, 56.4, 53.4, 51.0, 40.0.

Example 2(g)

Synthesis of(11aS)-8-Benzyloxy-7-methoxy-2-(4-methoxybenzylidene-1,2,3,11a,-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one(185) (See FIG. 14)

(2S)-N-[(2-allyloxycarbanylamino)-4-benzyloxy-5-methoxy]-2-(tertbutyldimethylsilyloxymethyl)-4-methylidenepyrrolidine (182)

The Wittig reagent, 4-methoxybenzylphosphonium bromide (3.686 g, 0.88mmol) was added portionwise to a suspension of sodium hydride (352 mg ofa 60% dispersion, 8.80 mmol) in anhydrous toluene (25 mL) under anitrogen atmosphere at 0° C. The mixture was allowed to warm to roomtemperature and then heated at reflux for 30 minutes. The colour of thereaction mixture darkened progressively from yellow through to orange.At this stage a solution of the ketone (6—see Example 1a) (0.5 g, 0.88mmol) in dry toluene (25 mL) was added dropwise to the reaction mixtureat reflux. After 10 minutes TLC (50% ethyl acetate, 50% 40-600 petroleumether) revealed the complete consumption of ketone. Excess toluene wasremoved by rotary evaporation under reduced pressure to yield a brownresidue, which was partitioned between ethyl acetate (100 mL) andsaturated sodium hydrogen carbonate (100 mL). The organic layer waswashed with brine (100 mL) and dried over magnesium sulphate) removal ofexcess solvent by rotary evaporation under reduced pressure gave a darkoil, which was subjected to flash chromatography on silica gel (20%ethyl acetate, 70% 40-60° petroleum ether). Removal of excess eluentafforded the product (182) as an oil which solidified on standing (420mg, 0.62 mmol, 71%). [α]²¹ _(D)−7.48° (c=1.002 CHCl₃). ¹H NMR (270 MHz,CDCl₃) cis/trans mixture, rotamers δ8.90 (bs, 1H), 7.95 (s, 1H),7.76-7.65 (m, 2H), 7.55 (m, 7H), 6.9 (s, 1H), 6.4) and 6.30 (2×bs, 1H),6.02-5.88 (m, 1H), 5.40-5.17 (s, 4H), 4.64-4.59 (m, 2H), 3.91-3.70 (m,9H), 3.00-2.95 (m, 2H). HRMS (FAB) 673 (M+1). Anal. Calcd forC₃₈H₄₈N₂O₇Si: C, 67.83; H, 7.19; N, 4.16. Found C, 67.64; H, 7.33; N,4.03.

(2S)-N-(2-allyloxycarnoylamino)-4-benzyloxy-5-methoxy]-2-(hydroxymethyl)-4-(4-methoxybenzylidene)pyrrolidine(183)

A solution of TBAF in THF (1.21 mL, 1 M solution, 1.21 mmol) was addedto a solution of 182 (0.65 g, 0.97 mmol) in THF (15 mL) at 0° C. Thereaction mixture was allowed to warm to room temperature and stirovernight. Excess THF was removed by rotary evaporation under reducedpressure and the residue was partitioned between ethyl acetate (100 mL)and saturated ammonium chloride (1 mL). The organic phase was washedwith brine (100 mL) and dried over magnesium sulphate. Excess solvent asevaporated under reduced pressure and the resulting residue wassubjected to flash column chromatography (silica gel, 50% ethyl acetateand 50% 40-60° petroleum ether). Removal of excess eluent by rotaryevaporation under reduced pressure afforded the compound 183 (0.9 g,1.61 mmol, 65%). ¹H NMR (270 MHz, CDCl₃) cis/trans mixture δ8.55 (bs,1H), 7.50-7.10 (m, 8H), 6.80-6.90 (m, 3H), 6.40 and 6.29 (2×bs, 1H),6.02-5.88 (m, 1H), 5.40-5.10 (m, 4H), 4.55-4.70 (m, 2H),4.50-4.30 (m,1), 3.95-3.80 (m, 8H), 3.10-3.90 (m, 1H), 3.50-3.70 (m, 1H). HRMS (FAB)Calcd for C₃₂H₃₅N₂O₇ (M+H) 559.2444; Found 559.2462.

(11S,11aS)-10-allyloxycarbonyl-8-benzyloxy-11-hydroxy-7-methoxy-2-(4-methoxybenzylidene)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one(184)

A solution of DMSO (0.41 mL, 5.80 mmol) in dry DCM (50 mL) was addeddropwise to a stirred solution of oxalyl chloride (1.45 ml of a 2Msolution, 2.90 mmol)at −40° C. under a nitrogen atmosphere. After 45minutes stirring at −45° C., a solution of 183 (0.9 g, 1.61 mmol) in DCM(50 mL) was added dropwise to the mixture over 45 minutes. Afterstirring at −45° C. for 45 minutes the reaction mixture was treateddropwise with a solution of TEA (0.94 mL, 6.76 mmol) in DCM (20 mL) over30 minutes. After a stirring at −45° C. for a further 40 minutes thereaction mixture was allowed to warm to room temperature and thendiluted with DCM (30 mL). The diluted reaction mixture was washed withdilute hydrochloric acid (1 N, 300 mL), water (150 mL), brine (150 mL)and dried over magnesium sulphate. Removal of excess solvent affordedthe crude product, which was subjected to column chromatography (silicagel, 50% ethyl acetate and 50% 40-60° petroleum ether). Removal ofexcess eluent afforded the product 184 as an oil (0.62 g, 1.11 mmol,69%). ¹H NMR (270 MHz, CDCl₃) cis/trans mix δ7.50-7.10 (m, 8H),6.90-6.85 (m, 2H), 6.74 (s, 1H), 6.50 and 6.45 (2×bs, 1H), 6.70-5.00 (m,6H), 4.70-4.20 (m, 4H), 3.98 (s, 3H), 3.90-3.70 (m, 4H), 3.10-2.80 (m,2H). HRMS (FAB) Calcd for C₃₂H₃₃N₂O₇ (M+H) 557.2288; Found 559.2277.

(11aS)-8-Benzyloxy-7-methoxy-2-(4-methoxybenzylidene-1,2,3,11a,-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one(185)

Triphenylphosphine, pyrrolidine and palladium tetrakistriphenylphosphinewere addaed sequentially to a stirred solution of substrate in dry DCM.The reaction mixture was allowed to stir at room temperature under anitrogen atmosphere for 2 h, at which time TLC (50% ethyl acetate and50% 40-60° petroleum ether) revealed the complete consumption ofstarting material. The reaction mixture was evaporated to dryness andthe resulting residue subjected to gravity column chromatography (silicagel, gradient elution: 30% ethyl acetate, 70% 40-600 petroleum ether to70% ethyl acetate, 30% 40-60° petroleum ether). Removal of excess eluentafforded the PBD (185) as a yellow glass that was reprecipitated fromethyl acetate with 40-60° petroleum ether.

¹H NMR (270 MHz, CDCl₃) cis/trans mix δ7.69 (d, 1H, J=4.39 Hz), 7.52 (s,1H), 7.46-7.30 (m, 5H), 7.20-7.16 (m, 2H), 6.92-6.88 (m, 2H), 6.84 (s,1H), 6.53 (bs, 1H), 5.20-5.17 (m, 2H), 4.52 (m, 2H), 3.96 (s, 3H),3.90-3.75 (m, 4H), 3.34-3.26 (m, 1H), 3.12-3.00 (m, 1H).

Example 3

Synthesis of Compounds of formula III

Overview of Synthesis

The Biaryl PBDs 136, 138 and 140 were obtained by removal of the Trocprotecting group from the protected carbinolamines 135, 137 and 139. Forcompounds 136 and 138 the deprotection method of Dong et al, wasemployed (Cd/Pb, ammonium acetate buffer), however, this approach couldnot be applied to the preparation of 140 as this molecule contained anitro group sensitive to the Cd/Pb couple. In this case a noveldeprotection procedure involving the use of tetrabutyl ammonium fluoridewas used. The protected biaryl carbinolamines were prepared by theSuzuki reaction, the common 7-iodo substituted protected carbinolamine134 was exposed to the appropriate boronic acid in the presence of apalladium catalyst. This reaction is of wide scope as over 70 boronicacids are commercially available. The iodo substituted protectedcarbinolamine 134 was furnished by Swern oxidation of the primaryalcohol 133. The Swern procedure was particularly effective in this casebut other oxidizing agents such as the Dess-Martin reagent, TPAP orpyridine sulphur trioxide complex and DMSO could also be employed. Theprimary alcohol 133 was afforded by coupling commercially availablepyrrolidinemethanol to the Troc protected anthranilic acid chlorideobtained by 132 by treatment with oxalyl chloride. The Troc protectedacid was in turn prepared by exposing the anthranilic acid 131 to2,2,2-trichloroethyl chloroformate. Other protecting groups can be usedin place of Troc such as Nvoc, Teoc and Fmoc but care must be taken inchoosing a protecting group as some groups such as Boc spontaneouslyform the isatoic anhydride when exposed to oxalyl chloride prior to thecoupling step.

The 9-methoxy PBD (101) was prepared in an analogous fashiondemonstrating the versatility of the approach.

The 8-amino PBD (151) was prepared by the removal of a Troc protectinggroup from the amino substituted protected carbinolamine 150. The freeamine was obtained by removal of an Fmoc protecting group under standardconditions (piperidine/DMF) from the protected carbinolamine 149. Swernoxidation of the primary alcohol 148 furnished 149 in good yield, thesubstrate for oxidation reaction was prepared by Fmoc protection of theaniline 147. Reduction of the nitro compound 146, with tin chloridefurnished the aniline, hydrogenation could not be employed to reduce thenitro group as the Troc system does not withstand these conditions. Thenitro compound 146 was prepared by the coupling of the acid chloridederived from 145 with pyrrolidinemethanol in the presence of base.Finally, the protected anthranilic acid 145 was furnished by exposingthe commercially available 4 nitro anthranilic acid 144 to TrocChloroformate.

The 8-benzyloxy-7,9-dimethoxy PBD (143, UP2022) was prepared by aslightly different approach which does not involve the use ofanthranilic acid starting materials but proceeds through 2-nitrobenzoicacid intermediates. The PBD was obtained from the protectedcarbinolamine 142 by removal of the Troc protecting group under theusual conditions. The protected carbinolamine was furnished by Swernoxidation of primary alcohol 141 which in turn was prepared by selectiveprotection of the amino alcohol 126 as the Troc carbamate by exposure toTroc Chloroformate in the presence of pyridine. The amino alcohol wasobtained by reduction of the nitro compound 125 with Raney Nickel andhydrazine (again hydrogenation could not be employed due to the presenceof a benzyl group). The nitro alcohol 125 was prepared by couplingpyrrolidine methanol to the requisite 2-nitrobenzoic acid 124. Thisnitro benzoic acid was not commercially available and was prepared infour steps from the available syringic acid 87. Nitration of the ester122 was proceeded smoothly using Copper nitrate in acetic anhydride. Theester 122 was obtained by standard methods.

The PBDs 96, 113, 120 and 194 were obtained in an identical fashion fromthe 2-nitrobenzoic acids 19, 108, 115 and 186.

The dimer 90 was prepared in an analogous fashion from the core nitrocompound 85; the core was assembled by joining together two units of thephenol 84 via mitsonobu etherification. The phenol 84 was derived fromsyringic acid 83 in a three step synthesis, the crucial step being thenitration of 82 which was performed with 70% nitric acid.

The phenolic PBD 130 was prepared by an analogous route to that used forthe synthesis of the PBD 143, however the requirement to incorporate aphenolic group prompted the use of a different protecting group, Teoc.The free PBD was obtained by treating the Teoc protected carbinolamine129 with TBAF in warm acetonitrile. The phenol 129 was unmasked by thehydrogenolysis of the benzyloxy moiety of 128 in the presence of theTeoc protecting group (Troc would not survive under these conditions).The benzyloxy compound 128 was obtained by Swern oxidation of theprimary alcohol 127 which was prepared by treating the amino alcohol 126with Teoc chloroformate in the presence of base.

Example 3(a)

Synthesis of the C9/C9′-Dimethoxy PBD Dimer (90, DRH-165) (See FIG. 15)

O-Acetylsyringic Acid (82)

A suspension of syringic acid 81 (10.0 g, 50.5 mmol) in acetic anhydride(30.0 g, 27.7 mL, 294.1 mmol) was warmed gently until a clear solutionwas obtained. Fused sodium acetate (0.5 g, 6.10 mmol) was added to thesolution which was allowed to stir for 16 hours at room temperature. Thesolution was poured into water (100 mL) and stirred thoroughly to ensurehydrolysis of any excess anhydride. Crude O-Acetyl-syringic acid wasrecrystallized from water to afford the product as an off-white powder(11.2 g, 46.7 mmol). H¹ NMR (270 MHz, CDCl₃) δ7.36 (s, 2H), 5.94 (br s,1H), 3.87 (s, 6H), 2.35 (s, 3H). HRMS calcd for 240.0634, found 240.0637

4-Acetoxy-3,5-dimethoxy-2-nitrobenzoic Acid (83)

Fuming nitric acid (5.2 mL) was added, carefully, to a solution ofo-acetylsyringic acid 82 (11.1 g, 46.2 mmol) in acetic anhydride (33 g,mmol) at 5° C. and the reaction mixture was then allowed to stir for 3hours at room temperature. The reaction mixture was poured over ice (300mL) and the yellow precipitate was collected by filtration, washed withwater (3×100 mL) and dried in vacuo to afford the product as a paleyellow solid (12.4 g). H¹ NMR (270 MHz, CDCl₃) δ7.37 (s, 1H), 3.92 (s,3H), 3.90 (s, 3H), 2.39 (s, 3H).

Methyl 3,5-dimethoxy-4-hydroxy-2-nitrobenzoate (84)

A catalytic amount of DMF (5 drops) was added to a solution of oxalylchloride (6.3 g, 49.8 mmol) and o-nitrobenzoic acid 83 (12.4 g, 45.2mmol) in anhydrous THF (100 mL) and the reaction mixture allowed to stirat room temperature for 16 h. The resulting acid chloride was quencheddropwise with anhydrous methanol (100 mL) at 0° C. The reaction mixturewas treated with potassium carbonate and allowed to stir at roomtemperature for 3 h. Excess solvent was removed by rotary evaporation atreduced pressure and the residue dissolved in water. The aqueoussolution was acidified to pH 8 and the resulting white precipitate wascollected by filtration, washed with water (2×100 mL) and dried toafford the product as an off-white solid (10.6 g, 83%). H¹ NMR (270 MHz,CDCl₃) δ10.07 (br s, 1H), 7.26 (s, 1H), 3.97 (s, 3H), 3.91 (s, 3H), 3.85(s, 3H).

1′,3′-Bis(4-carboxy-2,6-dimethoxy-5-nitrophenoxy)propane (85)

Diethylazidodicarboxylate (7.19 g, 41.3 mmol) was added dropwise over0.5 hours to a cooled, stirred solution of the phenol 84 (10.61 g, 41.3mmol) and TPP (16.24 g, 61.9 mmol) in anhydrous THF (100 mL), andallowed to stir for 1 h. A solution of 1,3-propanediol (1.57 g, 20.6mmol) in THF (30 mL) was added dropwise and the reaction mixture allowedto stir for 16 h. The reaction mixture was then treated with 1N aqueousNaOH (200 mL) and heated at reflux for 3 h. Excess solvent was removedby rotary evaporation under reduced pressure to afford an aqueoussuspension which was extracted with EtOAc (3×300 mL). The aqueousextract was acidified with concentrated HCl and the precipitatecollected by vacuum filtration. The precipitate was suspended in water(500 mL) and after stirring for 10 minutes, the suspension was filteredto afford the product as an orange solid (6.11 g, 60%). H¹ NMR (270 MHz,CDCl₃) δ7.32 (s, 2H), 4.36 (t, 4H,), 3.92 (s, 6H) 3.90 (s, 6H), 2.20 (t,2H).

(2S)-1,1′-[[(propane-1,3-diyl)dioxy]bis[2-nitro-3,5-dimethoxy-1,4-phenylene)carbonyl]]bis[2-(hydroxymethylpyrrolidine](86)

A catalytic amount of DMF (3 drops) was added to a solution of the acid85 (6.1 g, 12.4 mmol) and oxalyl chloride (2.37 mL, 3.45 g, 27.2 mmol)in anhydrous DCM (60 mL) and the reaction mixture allowed to stir atroom temperature for 16 h. The resulting acid chloride was addeddropwise over 0.5 hours to a stirred solution of TEA (6.26 g, 61.8 mmol)and pyrrolidinemethanol (2.75 g, 27.2 mmol) in anhydrous DCM (60 mL) at−10° C. The reaction mixture was then allowed to stir at roomtemperature for 6 h. The reaction mixture was washed with 1N HCl (3×100mL), water (3×100 mL), saturated NaHCO₃ (3×100 mL), brine (3×100 mL) anddried over MgSO₄. Removal of excess solvent by rotary evaporation underreduced pressure afforded the product as a yellow glass (8.25 g, 11.9mmol). H¹ NMR (270 MHz, CDCl₃) δ6.66 (s, 2H), 4.32-4.26 (m, 6H), 3.98(s, 6H), 3.90 (s, 6H), 3.86-3.67 (m, 4H), 3.41-3.27 (m, 4H), 2.23-2.12(m, 2H), 2.11-1.72 (m, 8H).

(2S)-1,1′-[[(propane-1,3-diyl)dioxy]bis[2-amino-3,5-dimethoxy-1,4-phenylene)carbonyl]]bis[2-(hydroxymethylpyrrolidine](87)

Hydrazine (3.45 g, 107.9 mmol) was added dropwise to a solution of 86 (1g, 1.45 mmol) in anhydrous methanol (40 mL) heated at reflux over Raneynickel (5 g, slurry). Heating was continued for a further 3 hours afterwhich time the reaction mixture was allowed to cool and filtered throughcelite to remove excess Raney nickel. The filtrate was evaporated todryness and dissolved in DCM (200 mL) and the organic solution washedwith water (2×100 mL), brine (2×100 mL) and dried over MgSO₄. Filtrationand evaporation of excess solvent in vacuo afforded the product as apink glass (5.59 g, 8.9 mmol, 98%). H¹ NMR (270 MHz, CDCl₃) δ6.54 (s,2H), 4.35 (br s, 2H), 4.29 (t, 4H), 3.85 (s, 3H), 3.83-3.46 (m, 14H),2.20-2.13 (m, 2H), 1.97-1.66 (m, 8H).

(2S)-1,1′-[[(propane-1,3-diyl)dioxy]bis[2-(2′,2′,2′-trichloroethoxycarbonyl)amino-3,5-methoxy-1,4-phenylene)carbonyl]]bis[2-(hydroxymethylpyrrolidine](88)

A solution of 2,2,2-trichloroethylchloroformate (1.45 g, 6.86 mmol, 1.9eq) in dry DCM (10 mL) was added dropwise over the space of 0.5 hours toa solution of 87 (2.28 g, 3.6 mmol) and pyridine (1.14 g, 14.4 mmol, 4eq) in dry DCM (50 mL) and allowed to stir for 16 hours at roomtemperature. The reaction mixture was diluted with DCM (200 mL) andwashed with 1N HCl (3×200 mL), H₂O (3×200 mL), brine (2×300 mL) anddried over anhydrous MgSO₄. Purification by flash chromatography (silicagel, EtOAc) afforded the product as a pale yellow glass (1.43 g). H¹ NMR(270 MHz, CDCl₃) Rotamers δ9.21 and 8.40 (2×br s, 2H), 6.49 and 6.54(2×s, 2H), 5.08-3.59 (m, 26H), 3.33-3.30 (m, 4H), 2.04-1.69 (m, 10H).

1,1′-[[Propane-1,3-diyl)dioxy]bis[(11S,11aS)-10-(2′,2′,2′-trichloroethoxycarbonyl)-11-hydroxy-7,9-dimethoxy-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one.(89)

A solution of dry DMSO (14.9 mmol, 1.17 g, 1.06 mL) in dry DCM (5 mL)was added dropwise over 20 minutes to a stirred solution of oxalylchloride in DCM (7.38 mmol, 3.69 mL of a 2N solution in DCM) under anitrogen atmosphere at −45° C. After stirring for an additional 15minutes, a solution of 88 (2.58 g, 2.63 mmol) in dry DCM (5 mL) wasadded dropwise over 45 minutes at −45° C. and stirred for 45 minutes at−45° C. TEA (2.12 g, 21.0 mmol) was added dropwise over 30 minutes andstirred for a further 15 minutes. The reaction mixture was allowed towarm to room temperature, and diluted with water (100 mL). The organiclayer was washed with 1N HCl (3×100 mL), water (3×100 mL), brine (3×100mL) and dried over anhydrous MgSO₄. Filtration and evaporation of thesolvent in vacuo afforded the product as a yellow glass (0.73 g). H¹ NMR(270 MHz, CDCl₃) δ7.06 (s, 2H), 5.61 (dd, 2H, J=3.39, 9.9 Hz), 4.74 (d,2H, J=11.72 Hz), 4.62 (d, 2H, J=11.91 Hz), 4.29-4.21 (m, 6H), 3.97-3.46(m, 16H), 2.28-2.01 (m, 10H).

Preparation of 10% Cd/Pb Couple

Yellow lead oxide (litharge, 1.8 g, 4.9 mmol) was dissolved in warm 50%aq. AcOH (50 mL) and the solution was slowly added to a vigorouslystirred suspension of Cd dust (Aldrich, 100 mesh, 5.46 g, 49 mmol) indeionised water (100 mL). The Cd darkened as Pb deposited on itssurface, and formed clumps that were gently broken up with a glass rod.The dark non-pyrophoric Cd/Pb couple was filtered, washed with water,acetone, crushed and dried prior to storage and use.

1,1′-[[Propane-1,3-diyl)dioxy]bis[(11aS)-7,9-dimethoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one.(90)

Cadmium/lead couple (3.8 mmol Cd, 0.47 g of C\Pb couple) was added to avigorously stirred solution of 89 (0.76 g, 0.8 mmol) in THF (10 mL) and1N NH₄OAc (10 mL) and stirring continued for 2.5 h. The reaction mixturewas diluted with DCM (150 mL) and dried over MgSO₄. Filtration andevaporation of the solvent in vacuo afforded the product as a yellowglass (0.32 g, 0.55 mmol, 71%). H¹ NMR (270 MHz, CDCl₃) mixture ofC11/C11′R/S carbinolamines δ7.08 (s, 2H), 5.53 (br s, 2H), 5.38 (br s,2H), 4.90 (d, 2H, J=9 Hz), 4.79 (d, 2H, J=9 Hz), 4.38-3.54 (m, 22H),2.27-1.79 (m, 10H). MS (FAB) m/e (relative intensity) 594 (M+2, 27%),593 (M+1, 69%)

Example 3(b)

Synthesis of the C7-Methoxy PBD (96, DRH-271) (See FIG. 16)

N-(3-Methoxy-2-nitrobenzoyl)pyrrolidin-2-methanol (92)

A catalytic amount of DMF (2 drops) was added to a stirred solution of3-methoxy-2-nitro-benzoic acid 91 (5.01 g, 25.4 mmol) and oxalylchloride (3.54 g, 27.9 mmol) in dry CHCl₂(50 mL) under a nitrogenatmosphere. The reaction mixture was allowed to stir overnight, beforebeing used directly in the preparation of 92. A solution of the acidchloride in anhydrous CHCl₂(50 mL) was added dropwise over 1 hour to avigorously stirred solution of pyrrolidinemethanol (2.57 g, 25.4 mmol)and TEA (6.42 g, 63.6 mmol) in anhydrous CHCl₂(50 mL) under a nitrogenatmosphere at 0° C. and allowed to stir overnight at room temperature.The reaction mixture was washed with 1N HCl (1×100 mL), H₂O (3×100 mL)and brine (3×100 mL). The organic layer was dried over anhydrous MgSO₄,and evaporation of the solvent afforded a brown oil (6.37 g, 22.7 mmol,89%).

N-(2-Amino-3-Methoxybenzoyl)pyrrolidin-2-methanol (93)

Hydrazine hydrate (4.37 g, 136.4 mmol) was added dropwise to a solutionof 92 (6.37 g, 22.7 mmol) in gently refluxing methanol (100 mL) overRaney nickel (2.4 g, slurry). The resulting vigorous evolution ofhydrogen gas subsided after approximately 10 minutes and the reactionwas deemed to be complete by TLC after 2 h. The reaction mixture wasfiltered through celite and the solvent evaporated. Distilled water (100mL) was added to the residue, and the aqueous mixture was extracted withEtOAc (3×100 mL) and washed with H₂O (3×100 mL) and brine (3×100 mL) anddried over anhydrous MgSO₄. Evaporation of the solvent afforded a brownglass (5.49 g, 21.8 mmol) as a single spot by TLC.

N-(3-Methoxy-2-((2′,2′,2′-trichloroethoxy)carbonylaminobenzoyl)pyrrolidin-2-methanol(94)

A solution of 2,2,2-trichloroethyl chloroformate (4.61 g, 21.8 mmol) indistilled dichloromethane (50 mL) was added dropwise over 0.5 hours to astirred solution of the substrate, 93 (5.46 g, 21.8 mmol) and anhydrouspyridine (3.44 g, 43.5 mmol) in distilled dichloromethane (100 mL) at 0°C. The reaction mixture was allowed to stir for 2.5 hours at which timeTLC showed reaction to be complete. The reaction mixture was dilutedwith anhydrous DCM (100 mL) and washed with 1N HCl (2×200 mL), H₂O (200mL), brine (200 mL) and dried over anhydrous MgSO₄. Evaporation of thesolvent afforded a brown oil which was purified by flash columnchromatography eluting with EtOAc to afford the product as a yellowsolid (6.14 g, 14.4 mmol); ¹H NMR (270 MHz, CDCl₃) δ1.75-2.25 (m, 4H),3.4-3.75 (m, 2H), 3.8 (s, 3H), 3.85-4.2 (m, 2H), 4.40 (m, 1H), 4.73-4.86(m, 2H), 6.86-6.97 (m, 2H), 7.85 (br d, 1H, J=9 Hz); ¹³C NMR (67.8 MHz,CDCl₃) δ169.9, 155.6, 152.4, 128.2, 127.8, 123.6, 116.0, 113.0, 95.4,74.4, 65.9, 60.9, 55.7, 51.0, 28.3, 24.9.

(11S,11aS)-10-(2′,2′,2′-trichloroethoxy)carbonyl-7-methoxy-11-hydroxy-1,2,3,10,11,-11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(95)

Anhydrous DMSO (3.14 g, 40.2 mmol) in dry DCM (25 mL) was added dropwiseover 5 minutes to a stirred solution of oxalyl chloride (2.53 g, 9.96 mLof a 2 N solution in DCM) under a nitrogen atmosphere at −50° C. Afterstirring for 5 minutes, the substrate 94 (6.03 g, 14.2 mmol) in dry DCM(25 mL) was added dropwise over 45 minutes to the reaction mixture,which was then allowed to stir for a further 45 minutes at −50° C. afterthe addition of the substrate. Dry TEA (5.72 g, 56.64 mmol) was addeddropwise to the mixture over 0.5 hours and the reaction mixture allowedto stir for a further 15 minutes. The reaction mixture was left to warmto room temperature and diluted with H₂O (100 mL). The organic phase waswashed with 1N HCl (2×200 mL), H₂O (2×200 mL), brine (2×200 mL) anddried over anhydrous MgSO₄. The solvent was evaporated to afford ayellow oil (6.68 g). The oil was subjected to flash chromatography withEtOAc as eluent to afford the product as a yellow solid (5.87 g, 13.9mmol); ¹H NMR (270 MHz, CDCl₃) δ1.99-2.14 (m, 4H), 3.45-3.77 (m, 2H),3.85 (s, 3H), 4.19 (br s, 1H), 4.28 (d, 1H, J=11.91 Hz), 5.14 (d, 1H,J=11.91 Hz), 5.66 (d, 1H, J=9.71 Hz), 6.97-7.02 (m, 1H), 7.23-7.27 (m,2H); ¹³C NMR (67.8 MHz, CDCl₃) d 166.8, 159.1, 154.7, 134.3, 131.5,129.9, 126.6, 118.106, 112.5, 112.3, 95.0, 86.0, 75.2, 75.1, 59.8, 55.7,46.7, 46.4, 28.7, 23.0, 21.0, 14.2.

7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(96)

10% Cd/Pb couple (2.50 g, 20 mmol Cd) was added to a rapidly stirringsolution of 95 (1.71 g, 4.03 mmol) in a mixture of THF (30 mL) and 1NNH₄₀Ac (30 mL). Upon addition, the solution turned cloudy and after 2hours TLC showed the reaction to be complete. The reaction mixture wasdiluted with EtOAc (150 mL) and dried over anhydrous MgSO₄. The solidswere filtered and rinsed with EtOAc (50 mL). Removal of excess solventby rotary evaporation under reduced pressure afforded the product as ayellow solid (0.84 g, 3.6 mmol, 90%)

Example 3(c)

Synthesis of the C7-Methoxy PBD (101, AG/140)(See FIG. 17)

3-methoxy-2-(2′,2′,2′-trichloroethoxycarbonylamino)benzoic acid (98)

2-amino-3-methoxybenzoic acid 97 (1 g, 6.0 mmol) and pyridine (0.97 mL,12.0 mmol) were dissolved in dry dichloromethane (30 mL). The resultingmixture was cooled and Troc-Cl (0.9 mL, 6.6 mmol) was added drop wise.The reaction mixture was allowed to stir overnight at room temperature,then washed with HCl (1N, 50 mL), water (50 mL) and brine (50 mL). Theorganic phase was dried over MgSO₄ and evaporated to yield 1.42 g ofcrude product, which was used in the next step without furtherpurification.

N-(3-methoxy-2-(2′,2′,2′-trichloroethoxycarbonylamino)benzoyl)-pyrrolidine-2-methanol(99)

Oxalyl chloride (0.57 mL, 6.58 mmol) together with 2 drops of dry DMFwas added to a solution of the crude product obtained from the previousreaction in dry dichloromethane (20 mL). After initial strongeffervescence, the mixture was allowed to stir at room temperatureovernight. The resulting acid chloride was added drop wise, over 30minutes to a solution of 2S-(+)-pyrrolidinemethanol (0.66 g, 6.58 mmol)and TEA (2.1 mL, 14.95 mmol) in dry dichloromethane (20 mL) at −16° C.Once coupling was complete the reaction mixture was diluted with ethylacetate (20 mL), and washed with 1N HCl (2×25 mL), satd. aqueous NaHCO₃(2×25 mL), water (25 mL) and brine (25 mL). The organic layer was thendried over MgSO₄ and evaporated to give a yellow oil. The crude productwas purified by flash chromatography (petroleum ether/ethyl acetate,50/50) to afford 0.54 g, of a pale yellow oil: ¹H NMR (270 MHz, CDCl₃) d1.6-1.8 (m, 1H); 1.81-2.0 (m, 2H); 2.02-2.21 (m, 1H); 3.4 (m, 1H); 3.6(m, 2H); 3.86 (m, 4H); 4.22 (dd, J=5.1, J=12.3 Hz, 1H); 4.72 (d, J=12Hz, 1H); 4.79 (d, J=12 Hz, 1H); 4.86 (m, 1H); 6.91 (s, 1H); 6.94 (s,1H); 7.2 (dd, J=7.5, J=8.4 Hz, 1H); 7.36 (bs, 1H). ¹³C NMR (67.8 MHz,CDCl₃) δ24.6; 28.8; 50.7; 55.9; 61.3; 66.5; 74.8; 75.3; 111.7; 111.9;119.1; 122.3; 126.3; 132.9; 152.7; 170.3 IR (Nujol): cm⁻¹ 3410, 2969,1738, 1613, 1583, 1514, 1429, 1268, 1218, 1109, 1079, 1049, 809, 759.MS: m/e (relative intensity) 425 (M+, 10), 394 (20), 323 (30), 276 (35),245 (100), 176 (100), 149 (45), 120 (40), 106 (20), 77 (30), 70 (100).HRMS Calculated for C₁₆H₁9C₁3N₂O₅:424.0357. Found: 424.0359. [α]²⁵_(D)=−45.1° (c=0.63, CHCl₃).

(11S,11aS)-11-hydroxy-9-methoxy-10-N-(2′,2′,2′-trichloroethoxycarbonyl)-1,2,-3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4] benzodiazepin-5-one (100)

A solution of DMSO (0.46 ml, 6.63 mmol) in of dry dichloromethane (10mL) was added drop wise over 30 minutes to a solution oxalyl chloride(3.30 mmol,) in dry dichloromethane (11.65 mL) at −40° C. The mixturewas allowed to stir for a further 30 minutes, a solution of 99 (1 g,2.37 mmol) in dichloromethane (15 mL) was then added drop wise over 1hour. Following the end of addition the mixture was allowed to stir at−45° C. for 60 minutes, then a solution of TEA (1.31 mL) indichloromethane (6 mL) was added drop wise and the mixture was allowedto warm to room temperature. The reaction mixture was washed with water(50 mL), 1N HCl (2×25 mL), satd. aqueous NaHCO₃(2×25 mL), and brine (50mL). The organic solution was dried over MgSO₄ and evaporated. The crudeproduct was purified by flash chromatography (silica gel EtOAc/petroleumether 1/1) to give a colourless oil (0.64 g, 63%): ¹H NMR (270 MHz,CDCl₃) δ2.01-2.15 (m, 4H); 3.43-3.58 (m, 2H); 3.73 (m, 2H); 3.83 (s,3H); 4.35 (d, J=12, 1H); 4.98 (d, J=12, 1H); 5.66 (dd, J=3.8, J=9.6 Hz,1H); 7.02 (dd, J=2.2, J=7.5 Hz, 1H); 7.35 (m, 2H). ¹³C NMR (67.8 MHz,CDCl₃) δ23.0; 28.6; 46.2; 56.1; 59.9; 75.3; 86.2; 94.8; 113.4; 120.2;123.1; 129.4; 134.9; 154.7; 155.4; 166.7. IR (Nujol): cm⁻¹ 3291, 2924,1724, 1616, 1580, 1463, 1318, 1278, 1075, 945, 812, 739. MS: m/e(relative intensity) 422 (M−1, 40), 387 (3), 275 (10), 245 (15), 217(10), 176 (100), 150 (8), 120 (6), 70 (95). HRMS Calculated forC₁₆H₁7Cl₃N₂O₅: 422.0202. Found: 422.0203. [α]²⁵ _(D)=+136.5° (c=0.19,CHCl₃).

(11aS)-9-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(101)

Finely ground Cd/Pb couple (1.02 g). was added in small portions to astirred solution of 100 (0.64 g, 1.51 mmol) in THF (10 mL) and 1M NH₄OAc(10 mL). The reaction was followed by TLC (EtOAc), when no more startingmaterial was observed, the mixture was poured into ethyl acetate (200mL). The organic phase was dried over MgSO₄ and evaporated to yield theproduct as a pale yellow oil (0.28 g, 80%): ¹H NMR (270 MHz, CDCl₃)δ2.15 (m, 4H); 3.52 (m, 2H); 3.87 (s, 3H); 5.15 (m, 1H); 6.8-7.2 (m,3H); 7.8 (d, J=4.7 Hz, 1H, imine H11). IR (Nujol): cm⁻¹ 3373, 2975,1621, 1576, 1440, 1419, 1250, 1075, 750. MS: m/e (relative intensity)230 (M^(+.), 100), 215 (45), 201 (20), 187 (5), 160 (5), 146 (4), 133(20), 105 (10), 76 (25), 70 (45), 63 (3), 51 (3). HRMS Calculated forC₁₃H₁₄N₂O₂: 230.1055. Found: 230.1055. [α]²⁵ _(D)=+455.3° (c=0.6,CHCl₃).

Example 3(d)

Synthesis of the 7,8-Dimethoxy PBD (106, AG/105) (See FIG. 18)

4,5-dimethoxy-2-(2′,2′,2′-trichloroethoxycarbonylamino)benzoic acid(103)

A solution of Troc-Cl (0.76 ml, 5.56 mmol) in dry dichloromethane (10mL) was added dropwise to 2-amino-4,5-dimethoxybenzoic acid 102 (1 g,5.1 mmol) and pyridine (0.82 ml, 10.1 mmol) in dry dichloromethane (20ml) at 0° C. The reaction mixture was allowed to stir overnight at roomtemperature and then washed with dilute HCl (1N, 2×25 ml), water (2×25ml) and brine (20 ml). The organic phase was dried over MgSO₄ andevaporated to yield of crude product (1.6 g), which was used in the nextstep without further purification.

N-(4,5-dimethoxy-2′-(2″,2″,2″-trichloroethoxycarbonylamino)benzoyl)-pyrrolidine-2-methanol(104)

Oxalyl chloride (0.38 mL, 4.33 mmol) was added to the crudeTroc-protected anthranilic acid, prepared in the previous reaction,together with 2 drops of dry DMF in dry dichloromethane (30 mL). Afterinitial strong effervescence, the mixture was allowed to stir at roomtemperature overnight. The resulting acid chloride was added dropwise,over 30 minutes, to a solution of 2S-(+)-pyrrolidinemethanol (0.44 g,4.33 mmol) and TEA (1.37 ml, 9.85 mmol) of dry dichloromethane (15 mL)at −16° C. The reaction mixture was diluted with ethyl acetate (20 mL),and washed with dilute HCl (1N, 2×30 mL), satd. aqueous NaHCO₃ (2×30mL), water (30 mL) and brine (30 mL). The organic layer was then driedover MgSO₄ and evaporated to give a yellow oil. The crude product waspurified by flash chromatography (petroleum ether/ethyl acetate=50/50)to yield the product (1.2 g, 70%) as a pale yellow oil: ¹H NMR (270 MHz,CDCl₃) δ1.75 (m, 2H); 1.92 (m, 1H); 2.17 (m, 1H); 3.53 (m, 2H); 3.72 (m,1H); 3.86 (s, 3H); 3.93 (s, 3H); 4.19 (m, 1H); 4.43 (m, 1H); 4.77 (d,J=12 Hz, 1H); 4.85 (d, J=12 Hz, 1H); 6.85 (s, 1H); 7.69 (s, 1H); 9.08(bs, 1H). ¹³C NMR (67.8 MHz, CDCl₃) δ25.1; 28.2; 51.4; 56.0; 56.4; 60.8;65.9; 74.4; 95.3; 104.7; 110.7; 116.3; 130.8; 144.4; 151.0; 152.1;170.4. MS: m/e (relative intensity) 454 (M−1, 5), 356 (3), 306 (10), 275(5), 206 (100), 179 (15), 150 (10), 136 (3), 70 (45). HRMS Calculatedfor C₁₇H₂₁C₁₃N₂O₆: 454.0465. Found: 454.0464. [α]²⁵ _(D)=−72.2° (c=0.18,CHCl₃).

(11s,11aS)-7,8-dimethoxy-11-hydroxy-10-N-(2′,2′,2′-trichloroethoxycarbonyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one (105)

A solution of DMSO (0.9 ml, 12.9 mmol) in dry dichloromethane (15 mL)was added dropwise over 30 minutes to a solution of oxalyl chloride (6.4mmol) of dry dichloromethane (15 mL) keeping the temperature below −40°C. The reaction mixture was allowed to stir for further a 30 minutes atwhich point a solution of 104 (2.1 g, 4.61 mmol) in dichloromethane (35mL) was added drop wise over 1 hour. After addition of the substrate thereaction mixture was allowed to stir at −45° C. for 60 minutes, and thentreated with a solution of TEA (2.56 mL) in of dichloromethane (10 mL)were added drop wise and the mixture was allowed to warm to roomtemperature. The reaction mixture was washed with water (75 mL), diluteHCl (1N, 75 mL), water (75 mL), brine (75 mL) dried over MgSO₄ andevaporated. The crude product was purified by flash chromatography(EtOAc/petroleum ether 40/60) to give a colourless oil (1.19 g, 57%): ¹HNMR (270 MHz, CDCl₃) δ2.04 (m, 2H); 2.11 (m, 2H); 3.47-3.59 (m, 2H);3.68-3.75 (m, 1H); 3.91 (s, 3H); 3.94 (s, 3H); 4.21 (d, J=12.1 Hz, 1H);4.43 (d, J=4.76 Hz, 1H); 5.27 (d, J=12.1 Hz, 1H); 5.65-5.7 (dd, J=4.58,J=9.71 Hz, 1H); 6.82 (s, 1H); 7.26 (s, 1H). ¹³C NMR (67.8 MHz, CDCl₃)δ23.1; 28.6; 46.4; 56.0; 56.1; 60.0; 74.9; 86.4; 95.1; 110.3; 112.7;125.6; 148.6; 150.8; 154.5; 167.0. MS: m/e (relative intensity) 452(M−1, 30), 424 (7), 354 (10), 276 (25), 206 (100), 180 (10), 150 (10),70 (100). HRMS Calculated for C₁₇H₁₉C₁₃N₂O₆: 452.0308. Found: 452.0309.[α]²⁵ _(D)=+104.7° (c=0.27, CHCl₃).

(11aS)-7,8-dimethoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(106, AG/105)

Finely ground Cd/Pb couple (3.12 g) was added portion wise to a solutionof 105 (1 g, 2.2 mmol) THF (10 mL) and NH₄OAc (1M, 10 mL). The reactionwas followed by TLC (EtOAc), when no starting material was present, themixture was poured into ethyl acetate (400 mL). The organic phase wasdried over MgSO₄ and evaporated to yield the crude product, which waspurified by flash chromatography (EtOAc) to give of the pure compound asa pale yellow oil (0.45 g, 78%): ¹H NMR (270 MHz, CDCl₃) δ2.08 (m, 2H);2.29 (m, 2H); 3.53-3.63 (m, 1H); 3.72 (m, 1H); 3.79-3.85 (m, 1H); 3.93(s, 3H); 3.96 (s, 3H); 6.82 (s, 1H); 7.52 (s, 1H); 7.68 (d, J=4.4, 1H).¹³C NMR (67.8 MHz, CDCl₃) δ24.2; 29.6; 46.7; 53.7; 56.0; 56.1; 109.4;111.2; 140.7; 147.5; 151.3; 162.5; 164.6. IR (Nujol): cm⁻¹ 3000-2800,1601, 1450, 1434, 1500, 1453, 1263, 1217, 1010, 908, 735. MS: m/e(relative intensity) 260 (M+, 100), 245 (50), 231 (25), 217 (10), 191(20), 164 (25), 136 (20), 121 (5), 93 (8), 70 (10). HRMS Calculated forC₁₄H₁₆N₂O₃: 260.1160. Found: 260.1161. [α]²⁵ _(D)=+1004.7° (c=0.17,CHCl₃).

Example 3(e)

Synthesis of the 6,7,8-Trimethoxy PBD (113, DRH-NA7) (See FIG. 19)

2,3,4-Trimethoxy-6-nitrobenzoic acid (108)

2,3,4-trimethoxybenzoic acid 107 (25 g, 117.8 mmol) was addedportionwise to a stirred solution of 70% nitric acid at 0° C. for 30minutes. The reaction mixture was poured into cold water (1250 mL) andstirring was continued for 30 minutes. The reaction mixture wasextracted with EtOAc (2×200 mL) and the combined organic layers werewashed with brine (2×200 mL) and dried over anhydrous MgSO4. Evaporationof excess solvent in vacuo afforded the product as a pure whitecrystalline solid (18.67 g, 60%): R_(f)=0.5 (silica, EtOAc); IR (nujol)2922, 1713, 1618, 1570, 1504, 1464, 1401, 1308, 1246, 1168, 1111, 1028,920, 852, 789, 773, 728, 689 cm⁻¹; ¹H NMR (270 MHz, CDCl₃) δ7.76 (1H,s), 4.0 (3H, s), 3.95 (3H, s), 3.90 (3H, s); ¹³C NMR (67.8 MHz, CDCl₃)δ166.0, 153.2, 150.1, 147.79, 139.6, 120.8, 103.6, 62.2, 61.1, 56.5; MS(EI) m/z 258 (M+1), 240, 214.

N-(2-Nitro-4,5,6-trimethoxybenzoyl)pyrrolidine-2-methanol (109)

A catalytic quantity of DMF (2 drops) was added to a stirred solution of108 (10 g, 38.9 mmol) and oxalyl chloride (5.87 g, 46.2 mmol) in dryCHCl₂ (100 mL) under a nitrogen atmosphere. The reaction mixture wasallowed to stir overnight, and the product was used directly in the nextstage of the reaction. The newly formed acid chloride was added dropwiseto a stirred solution of pyrrolidinemethanol (3.92 g, 38.8 mmol) andanhydrous triethylamine (12.4 mL, 9.8 g, 97.0 mmol) in anhydrous DCM (50mL) at 0° C. under nitrogen. Once the addition was complete, thereaction mixture was left to warm to room temperature and left to stirovernight. The reaction mixture was washed with 1N HCl (100 mL), water(100 mL), and brine (2×100 mL). The combined organic layers were dried(MgSO₄) and the solvent was removed in vacuo to afford 109 (12.1 g, 91%)as a pale yellow oil: R_(f)=0.39 (silica, EtOAc); [α]^(21.9) _(D)+135°(c=0.1, DCM); IR (neat) 3400, 3105, 2947, 2878, 1652, 1568, 1538, 1455,1348, 1250, 1195, 1115, 975, 922, 849, 822, 792, 758, 733, 646 cm⁻¹; ¹HNMR (270 MHz, CDCl₃) δ7.59 (1H, s), 4.46 (2H, d, J=2.93 Hz), 4.07 (3H,s), 4.03 (3H, s), 4.01 (3H, s), 3.89 (3H, t), 3.45-3.29 (2H, m),2.24-2.17 (2H, m), 2.00-1.84 (2H, m); ¹³C NMR (67.8 MHz, CDCl₃,rotamers) δ165.7, 165.1, 153.3, 149.2, 148.1, 138.8, 122.5, 104.1, 66.4,65.5, 62.4, 62.3, 61.3, 56.6, 49.2, 49.0, 28.7 24.3; MS (EI) m/z 341(M+1), 324, 309, 293, 277, 264, 254.

N-(2-Amino-4,5,6-trimethoxybenzoyl)pyrrolidine methanol (110)

Hydrazine hydrate (5.67 g, 177.2 mmol) was added dropwise to a solutionof 109 (12.1 g, 35.47 mmol) in gently refluxing methanol (142 mL) overRaney nickel (3.45 g, slurry). The resulting vigorous evolution ofhydrogen gas subsided after approximately 10 minutes and the reactionwas deemed to be complete by TLC after 3 h. The reaction mixture wasfiltered through celite and the solvent evaporated. Distilled water (200mL) was added to the residue, and the aqueous mixture was extracted withDCM (2×100 mL) and the combined organic phase washed with H₂O (3×100 mL)and brine (3×100 mL) and dried over anhydrous MgSO₄. Evaporation of thesolvent afforded 110 (11.24 g) as a yellow oil. R_(f)=0.14 (silica,EtOAc); [α]^(21.8) _(D)=+100° (c=0.1, DCM); IR (neat) cm⁻¹ 3355, 2940,2879, 2843, 1614, 1498, 1463, 1428, 1410, 1365, 1339, 1240, 1199, 1123,1078, 1039, 997, 915, 817, 731, 646; ¹H NMR (270 MHz, CDCl₃) δ6.10 (1H,s), 4.37 (2H, d, J=3.67 Hz), 3.93 (3H, s), 3.88 (3H, s), 3.86 (3H, s),3.67 (2H, t), 2.17-2.02 (2H, m), 1.87-1.82 (2H, m) ¹³C NMR (67.8 MHz,CDCl₃) δ168.8, 154.7, 150.9, 149.6, 140.6, 133.8, 95.8, 66.5, 61.8,61.4, 61.3, 61.1, 49.2, 28.6, 24.4; MS (EI) m/z 310 (M⁺), 294, 279, 229,210, 194, 180, 149, 124, 102, 83, 70, 57.

N-(2-[2′,2′,2′-Trichloroethoxycarbonylamino]-4,5,6-trimethoxybenzoyl)pyrrolidine-2-methanol(111)

A stirred solution of 110 (11.24 g, 36.3 mmol) in DCM (150 mL) andpyridine (5.86 mL, 5.73 g, 72.5 mmol) was treated dropwise with2,2,2-trichloroethyl chloroformate (5 mL, 7.61 g, 35.9 mmol) in DCM (50mL) under a nitrogen atmosphere at 0° C. One hour after the addition of2,2,2-trichloroethyl chloroformate, the reaction mixture was dilutedwith DCM (100 mL) and washed with 1N HCl (100 mL), water (2×150 mL),brine (2×100 mL) and dried (MgSO₄). The solvent was removed in vacuo toafford 111 (15.44 g, 88%) as a clear brown oil: R_(f)=0.44 (silica,EtOAc); IR (neat) cm⁻¹ 3437, 2948, 1738, 1628, 1497, 1458, 1422, 1397,1238, 1115, 1027, 1008, 823, 760, 624; ¹H NMR (270 MHz, DMSO) δ6.82 (1H,s), 5.06 (2H, s), 4.04 (2H, d, J=6.83 Hz), 3.85 (3H, s), 3.84 (3H, s),3.79 (3H, s), 3.67 (2H, t), 2.00-1.97 (2H, m), 1.96-1.88 (2H, m) ¹³C NMR(67.8 MHz, DMSO) δ164.2, 153.5, 149.6, 139.6, 129.4, 121.3, 96.2, 73.9,61.4, 60.9, 58.7, 56.2, 47.9, 27.5, 23.7; HRMS (FAB) calcd forC₁₈H₂₃N₂O₇Cl₃ (M⁺) 484.0571, found 484.0944.

6,7,8-Trimethoxy-10-(2′,2′,2′-trichloroethoxycarbonyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(112)

A solution of oxalyl chloride in DCM (22.3 mL of a 2N solution, 44.7mmol) diluted with anhydrous DCM (42 mL) at −45° C. was treated dropwisewith a solution of anhydrous DMSO (6.39 mL, 90.2 mmol) in anhydrous DCM(16.24 mL) over a period of 15 minutes. The reaction mixture was stirredat −45° C. for 15 minutes and treated with a solution of 111 (15.44 g,31.7 mmol) in dry DCM (34.3 mL) and stirred at −45° C. for 45 minutes.Triethylamine (17.7 mL, 127.1 mmol) was added dropwise to the reactionmixture over 0.5 h, and then allowed to stir for a further 15 minutes.The reaction mixture was allowed to warm to room temperature and dilutedwith water (100 mL). The organic layer was washed with 1N HCl (200 mL),water (200 mL), brine (200 mL) and dried (MgSO₄). The reaction mixturewas evaporated and purified by flash column chromatography (EtOAc) toafford the product 112 (8.27 g, 54%) as a clear yellow glass: R_(f)=0.48(silica, EtOAc); [α]^(22.2) _(D)+190° (c 0.15, DCM); IR (neat) cm⁻¹3262, 2979, 2943, 2885, 1732, 1613, 1493, 1456, 1399, 1372, 1334, 1299,1264, 1244, 1201, 1118, 1059, 1014, 969, 926, 888, 838, 784, 756, 720,693, 624; ¹H NMR (270 MHz, CDCl₃) δ6.64 (1H, s), 5.58 (1H, s), 5.31 (1H,s), 4.34 (1H, d, J=19.78 Hz), 4.15-4.00 (1H, m), 3.95 (3H, s), 3.91 (3H,s), 3.90 (3H, s), 3.77 (2H, t), 3.55 (1H, t), 2.17-2.14 (2H, m),2.14-2.10 (2H, m). ¹³C NMR (67.8 MHz, CDCl₃) δ163.49, 154.32, 152.30,142.69, 129.51, 121.16, 109.35, 95.20, 85.63, 62.30, 61.36, 60.48,56.09, 45.56, 28.44, 22.85; MS (EI) m/z 485 (M+1), 467, 398, 384, 350,291, 254, 236, 222, 194, 131, 102, 82, 70, 57.

6,7,8-Trimethoxy-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(113)

10% Cd/Pb couple (2.57 g, 20.6 mmol Cd) was added to a stirred solutionof 112 (2.00 g, 4.1 mmol) in THF (20 mL) and 1N NH₄OAc buffer (20 mL)and left at room temperature for 4 h. The reaction mixture was dilutedwith EtOAc (200 mL) and washed with water (2×100 mL). The organic layerwas washed with brine (2×100 mL) and dried (MgSO₄). The solvent wasremoved in vacuo to give 113 (0.76 g, 64%) as a yellow glass: R_(f)=0.1(silica, EtOAc); [α]^(20.7) _(D)=+505° (c=0.1, DCM); IR (neat) cm⁻¹3339, 2976, 2939, 1614, 1455, 1428, 1392, 1359, 1275, 1245, 1203, 1113,1052, 1035, 1000, 926, 804, 751, 665; ¹H NMR (270 MHz, CDCl₃) δ (1H, d,J=4.39 Hz), 6.61 (1H, s), 6.14 (1H, d, J=8.24 Hz), 4.36 (1H, d, J=8.79Hz), 4.01 (3H, s), 3.98 (3H, s), 3.84 (3H, s), 3.48-3.46 (2H, m)2.26-2.23 (2H, m), 2.16-1.93 (2H, m); HRMS (FAB) calcd for C₁₅H₁₈N₂O₄(M+1) 290.1266, found 290.1208.

Example 3(f)

Synthesis of the 7,8,9-Trimethoxy PBD (120, DRH-69) (See FIG. 20)

3,4,5-Trimethoxy-2-nitrobenzoic acid (115)

Methyl 3,4,5-trimethoxy-2-nitrobenzoic 114 (24.37 g, 89.9 mmol) wasadded to a 5% solution of KOH (18 g) in MeOH (357 mL). The mixture washeated at reflux for 50 minutes. Evaporation of the solvent afforded agrey residue, which was dissolved in H₂O (200 mL) The resulting alkalinesolution was acidified to pH1 with concentrated HCl, and extracted withCHCl₃ (3×100 mL). The organic layer was washed with H₂O (3×100 mL),brine (3×100 mL) and dried over anhydrous MgSO₄. Filtration andevaporation of the solvent afforded a pure white crystalline solid(20.67 g, 80.4 mmol): ¹H NMR (270 MHz, CDCl₃) δ3.9 (s, 3H), 4.0 (s, 3H),4.1 (s, 3H), 7.4 (s, 1H), 12.4 (br s, 1H).

N-(2-Nitro-3,4,5-trimethoxybenzoyl)pyrrolidine-2-methanol (116)

A catalytic amount of DMF (2 drops) was added to a stirred solution of115 (2.57 g, 10 mmol) and oxalyl chloride (1.40 g, 11 mmol) in dryCH₂Cl₂ (40 mL) under an inert atmosphere. The reaction mixture wasallowed to stir overnight, the resulting solution of the acid chloride,(2.76 g, 10 mmol) in anhydrous CH₂Cl₂ (40 mL) was added dropwise over 1hour to a vigorously stirred solution of pyrrolidinemethanol (1.11 g, 11mmol) and TEA (2.52 g, 25 mmol) in anhydrous CH₂Cl₂ (40 mL) under anitrogen atmosphere at 0° C. and allowed to stir overnight at roomtemperature. The reaction mixture was washed with 1N HCl (1×50 mL), 1NNaOH (1×50 mL), H₂O (3×50 mL) and brine (3×50 mL) and dried overanhydrous MgSO₄. Filtration and evaporation of the solvent afforded ayellow oil (2.81 g, 8.3 mmol): Rf=0.47 (5% MeOH/CHCl₃); ¹H NMR (270 MHz,CDCl₃) δ1.7-2.0 (m, 3H), 2.1-2.2 (m, 1H), 3.3-3.5 (m, 2H), 3.7-3.9 (m,2H), 3.9-4.0 (2×s, 6H), 4.0-4.1 (s, 3H), 4.2-4.3 (m, 1H), 6.7 (s, 1H);¹³C NMR (67.8 MHz, CDCl₃) δ167.3, 156.5, 147.9, 143.5, 128.8, 104.8,65.8, 62.6, 61.4, 61.2, 56.6, 50.2, 28.4, 28.1, 24.5, 14.2.

N-(2-Amino-3,4,5-trimethoxybenzoyl)pyrrolidine-2-methanol (117)

Hydrazine hydrate (1.33 mL, 41.5 mmol) was added dropwise to a solutionof 116 (2.83 g, 8.3 mmol) in methanol (142 mL) gently refluxing overRaney nickel (500 mg, slurry). The resulting vigorous evolution ofhydrogen gas subsided after approximately 10 minutes and the reactionwas deemed to be complete by TLC after 2 h. The reaction mixture wasfiltered through celite and the solvent evaporated. Distilled water (100mL) was added to the residue, and the aqueous mixture was extracted withEtOAc (3×100 mL) and the combined organic phase washed with H₂O (3×100mL) and brine (3×100 mL) and dried over anhydrous MgSO₄. Evaporation ofthe solvent afforded the product (2.18 g, 6.5 mmol) as a brown oil: ¹HNMR (270 MHz, CDCl₃) δ1.6-2.0 (m, 3H), 2.1-2.2 (m, 1H), 3.4-3.7 (m, 4H),3.8 (s, 3H), 3.8-3.9 (2×s, 6H), 4.4 (br s, 1H), 4.7-4.3 (br s, 1H), 6.6(s, 1H); ¹³C NMR (67.8 MHz, CDCl₃) δ144.7, 144.5, 141.6, 134.6, 107.1,66.9, 61.0, 60.9, 60.5, 56.8, 50.9, 28.6, 24.9, 21.1, 14.2.

N-2-(Trichloroethoxycarbonylamino)-3,4,5-trimethoxybenzoyl)pyrrolidine-2-methanol(118)

A solution of 2,2,2-trichloroethylchloroformate (1.37 g, 6.5 mmol) indistilled dichloromethane (40 mL) was added dropwise over 0.5 hours to asolution of anhydrous pyridine (0.93 g, 11.8 mmol) and the substrate,117 (1.82 g, 5.9 mmol) in distilled dichloromethane (60 mL) at 0° C.After 1.5 h. the reaction mixture was diluted with anhydrous DCM (100mL) and washed with 1N HCl (2×100 mL), H₂O (100 mL), brine (100 mL) anddried over anhydrous MgSO₄. Evaporation of the solvent yielded a brownoil which was purified by flash column chromatography eluting with 1%MeOH/99% CHCl₃ to afford the product as a yellow oil (1.83 g, 3.8 mmol):¹H NMR (270 MHz, CDCl₃) δ1.6-1.9 (m, 3H), 2.1-2.2 (m, 1H), 3.3-3.6 (m,2H), 3.6-3.85 (m, 2H), 3.8-3.9 (m, 9H), 4.2-4.3 (m, 1H), 4.7-4.8 (br s,1H), 4.8 (s, 2H), 6.6 (s, 1H); ¹³C NMR (67.8 MHz, CDCl₃) δ169.9, 153.2,151.9, 143.1, 128.5, 120.1, 105.2, 95.3, 74.6, 66.3, 61.2, 61.2, 61.0,56.3, 50.6, 28.7, 24.6.

(11S,11aS)7,8,9-trimethoxy-11-hydroxy-10-N-(2′,2′,21-trichloroethoxycarbonyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(119)

Anhydrous DMSO (3.15 mL, 44.3 mmol) in dry DCM (8.2 mL) was addeddropwise over 20 minutes to a stirred solution of oxalyl chloride (2.79g, 11.0 mL of a 2N solution in DCN; 22.0 mmol) in dry DCM (20.6 mL)under an inert atmosphere at −45° C. (varied between −38° and −48° C.).After stirring for 15 minutes, the substrate (7.59 g ; 15.6 mmol) in dryDCM (17 mL) was added dropwise over 45 minutes to the reaction mixture,which was then stirred for a further 45 minutes at −45° C. after thefinal addition of the substrate. Dry TEA (4.84 g, 48.0 mmol, 4 eq) wasadded dropwise to the mixture over 0.5 hours and stirred for a further15 minutes. The reaction mixture was allowed to warm to room temperatureand the reaction mixture diluted with H₂O (80 mL). The organic phase wasseparated, washed with brine (2×100 mL) and dried over anhydrous MgSO₄.The solvent was evaporated to afford the product as an off-white solid(4.39 g, 9.1 mmol): ¹H NMR (270 MHz, CDCl₃) δ1.95-2.2 (m, 4H), 3.4-3.8(m, 2H), 3.8-3.9 (m, 9H), 4.05 (d, 1H), 4.5-4.8 (dd, 2H), 5.6-5.7 (q,1H), 7.1 (s, 1H); ¹³C NMR (CDCl₃) rotamers δ166.7, 166.5, 155.2, 153.5,153.3, 150.0, 144.5, 129.5, 129.0, 121.7, 106.4, 106.2, 94.6, 86.1,85.9, 75.7 75.2, 61.5, 61.3, 60.9, 60.1 59.8, 56.2, 56.1, 46.5, 46.3,28.7, 28.6, 23.0.

7,8,9-Trimethoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(120, DRH-69)

10% Cd/Pb couple (1.25 g, 10 mmol Cd) was added to a rapidly stirringsolution of the Troc-carbamate, 119 (1.00 g, 2.1 mmol) in a mixture ofTHF (13 mL) and 1N NH₄OAc (8 mL). Upon addition, the reaction mixturewent cloudy. After 40 minutes, TLC showed the reaction to be completeand the reaction mixture was diluted with EtOAc (200 mL). The solutionwas dried over anhydrous MgSO₄ and the solids were filtered and rinsedwith EtOAc (50 mL). Evaporation of the solvent yielded the product as ayellow glass (0.581 g, 2.0 mmol). ¹H NMR (270 MHz, CDCl₃) δ7.73 (d, 1H,J=4.57 Hz), 7.08 (s, 1H), 4.0-3.4 (m, 12H), 2.4-1.8 (m, 4H)

Example 3(g)

8-Hydroxy-7,9-dimethoxy-1,2,3,11a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepin-5-one(130, DRH-168)

Methyl 4-hydroxy-3,5-dimethoxybenzoate (121)

Concentrated sulphuric acid (3 mL), was added dropwise to a solution of81 (20.24 g, 102.1 mmol) in refluxing methanol (70 mL). The reactionmixture was heated at reflux for a further 5 hours and then cooled toroom temperature and concentrated to a third of its original volume. Theconcentrate was poured onto crushed ice (c. 150 ml)and allowed to standfor 30 minutes. The aqueous mixture was extracted with ethyl acetate(3×100 mL) and the combined organic phase washed with distilled water(3×100 mL), brine (3×100 mL) and dried over anhydrous MgSO₄. Removal ofecxess solvent under reduced pressure afforded the product as a yellowsolid, 121 (18.39 g, 86.7 mmol; ¹H NMR (270 MHz, CDCl₃) δ3.9 (s, 3H),3.95 (s, 3H), 3.975 (s, 3H), 6.1 (s, 1H), 7.3 (s, 2H); ¹³C NMR (67.8MHz, CDCl₃) 3 166.9, 146.6, 139.2, 121.0, 106.6, 56.4, 52.1.

Methyl 4-Benzyloxy-3,5-dimethoxybenzoate (122)

Benzyl chloride (11.04 g, 86.9 mmol) was added to a stirred solution of121 (19.22 g, 90.8 mmol) over K₂CO₃ (6.59 g, 47.7 mmol) in anhydrousMeOH (175 mL) and the mixture was heated at reflux for 12 h. Excesssolvent was removed under reduced pressure and the residue was extractedwith benzene (3×100 mL). The organic layer was washed with H₂O (3×100mL), brine (3×100 mL) and dried over anhydrous MgSO₄. Evaporation of thesolvent afforded an orange oil which crystallised on standing. The solidwas redissolved in EtOAc, and briefly washed with 1N NaOH (100 mL), H₂O(100 mL), brine (100 mL) and dried over MgSO₄. Evaporation of excesssolvent yielded the product as a yellow solid 122 (19.20 g, 63.6 mmol);¹H NMR (270 MHz, CDCl₃) δ3.8 (s, 3H), 3.85 (s, 3H), 3.9 (s, 3H), 5.1 (s,2H), 7.3-7.5 (m, 7H); ¹³C NMR (67.8 MHz, CDCl₃) δ166.7, 153.2, 140.8,137.3, 128.7, 128.6, 128.4, 128.4, 128.2, 128.0, 127.7, 125.3, 106.7,74.9, 56.1, 52.2.

Methyl 2-nitro-4-benzyloxy-3,5-dimethoxybenzoate (123)

Finely ground copper nitrate (Cu(NO₃)₂, 14.79 g, 78.7 mmol) was addedportionwise to a vigorously stirred solution of the substrate (19.00 g,62.9 mmol) in acetic anhydride (120 mL) whilst keeping the reactiontemperature below 40° C. The reaction mixture was stirred for 1 hour andthen poured over ice (800 mL). The aqueous mixture was left to stir for1 hour and the product collected by filtration to afford a yellow solid(18.7 g); ¹H NMR (270 MHz, CDCl₃) δ3.85 (s, 3H), 3.95 (s, 3H), 3.96 (s,3H), 5.19 (s, 2H), 7.3-7.5 (m, 6H); ¹³C NMR (67.8 MHz, CDCl₃) δ163.2,154.3, 146.0, 145.2, 136.2, 128.7, 128.5, 128.4, 128.3, 117.8, 108.52,75.5, 62.7, 56.5, 53.0.

2-Nitro-4-benzyloxy-3,5-dimethoxybenzoic acid (124)

Potassium hydroxide (10.84 g, 193.6 mmol) was added to a stirredsolution of the substrate (18.7 g, 53.9 mmol) in anhydrous methanol (220mL) and the reaction mixture heated at reflux for 2 h. The reactionmixture was allowed to cool and acidified to pH2 with 1N HCl andextracted with chloroform (3×100 mL). The combined organic layers werewashed with water (3×200 mL), brine (3×200 mL) and dried over MgSO₄.Evaporation of excess solvent by rotary evaporation under reducedpressure afforded the product as a yellow solid (17.01 g, 51.1 mmol,95%); ¹H NMR (270 MHz, CDCl₃) δ3.9 (br s, 3H), 3.9 (br s, 3H), 5.1 (brs, 2H), 7.2-7.5 (m, 6H).

N-(4-Benzyloxy-3,5-dimethoxy-2-nitrobenzoyl)pyrrolidine-2-methanol (125)

A catalytic amount of DMF (5 drops) was added to a stirred solution of124 (10 g, 30.0 mmol) and oxalyl chloride (4.65 g, 36.0 mmol) in dryCH₃CN (115 mL) under a nitrogen atmosphere. The reaction mixture wasallowed to stir overnight and the resulting acid chloride used directlyin the next part of the procedure.4-benzyloxy-3,5-dimethoxy-2-nitro-benzoyl chloride in anhydrous CH₃CN(115 mL) was added dropwise over 0.5 hours to a stirring solution ofpyrrolidine methanol (3.34 g, 33.03 mmol, 1.1 eq) and TEA (7.58 g, 75.1mmol, 2.5 eq) in anhydrous DCM (100 mL) at 0° C. under a nitrogenatmosphere and the reaction mixture was allowed to stir overnight atroom temperature. The reaction mixture was washed with 1N HCl (2×100mL), and the organic layer was washed with distilled H₂O (2×100 mL),brine (2×100 mL) and dried over anhydrous MgSO₄. Evaporation of thesolvent yielded a brown glass (8.71 g, 20.9 mmol, 70%); ¹H NMR (270 MHz,CDCl₃) δ1.7-2.2 (m, 4H), 3.3-3.5 (m, 2H), 3.7-3.9 (m, 2H), 3.9 (s, 3H),4.0 (s, 3H), 4.2-4.3 (m, 1H), 5.1 (s, 2H), 6.85 (s, 1H), 7.3-7.5 (m,5H); ¹³C NMR (67.8 MHz, CDCl₃) δ167.3, 156.8, 148.2, 142.3, 136.4,136.0, 129.0, 128.5, 128.4, 104.8, 75.6, 65.7, 62.8, 61.4, 56.6, 50.2,28.3, 24.5.

N-(2-Amino-4-Benzyloxy-3,5-dimethoxybenzoyl)pyrrolidine-2-methanol (126)

Hydrazine hydrate (2.31 g, 72.2 mmol) was added dropwise to a solutionof 125 (6.01 g, 14.4 mmol) in methanol (60 mL) gently refluxing overRaney nickel (1.1 g, slurry). The resulting vigorous evolution ofhydrogen gas subsided after approximately 10 minutes and the reactionwas deemed to be complete by TLC after 2 h. The reaction mixture wasfiltered through celite and the solvent evaporated. Distilled water (100mL) was added to the residue, and the aqueous mixture was extracted withEtOAc (3×100 mL) and the combined organic phase washed with H₂O (3×100mL) and brine (3×100 mL) and dried over anhydrous MgSO₄. Evaporation ofthe solvent afforded the produce as a brown oil (3.97 g, 10.3 mmol,73%): ¹H NMR (270 MHz, CDCl₃) δ1.6-2.2 (m, 4H), 3.5-3.8 (m, 4H), 3.8 (s,3H), 3.9 (s, 3H), 4.4 (br s, 1H), 5.1 (s, 2H), 6.6 (s, 1H), 7.3-7.6 (m,5H); ¹³C NMR (67.8 MHz, CDCl₃) δ171.5, 144.9, 143.5, 141.9, 137.5,134.6, 128.6, 128.5, 128.3, 128.2, 128.0, 115.1, 107.3, 75.1, 66.9,61.0, 60.6, 60.4, 56.9, 50.9, 28.5, 24.9, 21.1, 14.2.

N-(4-Benzyloxy-3,5-dimethoxy-2-[(2′-trimethylsilylethoxy)carbonylamino[benzoyl)pyrrolidine-2-methanol(127)

A solution of anhydrous pyridine (0.21 g, 2.6 mmol) in anhydrous DCM (10mL) was added dropwise over 15 minutes to a stirred solution of2-(trimethylsilyl)ethanol (0.92 g, 7.8 mmol) and triphosgene (0.77 g,2.6 mmol) in anhydrous DCM (30 mL). The reaction mixture was allowed tostir overnight and the resulting solution of 2-(trimethylsilyl)ethylchloroformate added dropwise over 0.5 hours to the amine 126 (1.98 g,5.1 mmol) and anhydrous pyridine (1.22 g, 15.4 mmol) in distilleddichloromethane (70 mL) at 0° C. The reaction mixture was allowed tostir overnight at room temperature, diluted with anhydrous DCM (100 mL),washed with 1N HCl (3×100 mL), H₂O (3×100 mL), brine (3×100 mL) anddried over anhydrous MgSO₄. Filtration and evaporation of the solventyielded the product as a colourless glass (1.43 g, 2.7 mmol, 53%); ¹HNMR (270 MHz, CDCl₃) δ−0.05 (s, 9H), 0.94-0.99 (m, 2H), 1.66-2.12 (m,4H), 3.32-3.54 (m, 2H), 3.74-3.88 (m, 8H), 4.05-4.22 (m, 3H), 4.69 (brs, 1H), 4.97 (s, 2H), 6.57 (s, 1H), 6.64 (br s, 1H), 7.23-7.43 (m, 5H);¹³C NMR (67.8 MHz, CDCl₃) δ170.1, 155.1, 151.4, 148.1, 142.0, 137.1,128.4, 128.3, 128.1, 121.2, 105.6, 75.3, 66.1, 64.0, 61.3, 61.0, 56.3,50.6, 28.7, 24.7, 17.6, −1.5.

(11S,11aS)-8-benzyloxy-7,9-dimethoxy-11-hydroxy-10-N-(2′-trimethylsilylethoxycarbonyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one. (128)

Anhydrous DMSO (0.57 g, 7.2 mmol) in dry DCM (5 mL) was added dropwiseover 30 minutes to a stirred solution of oxalyl chloride (0.46 g, 3.6mmol) in dry DCM (5 mL) under a nitrogen atmosphere at −45° C. Afterstirring for 15 minutes, the substrate (1.35 g, 2.6 mmol) in dry DCM (15mL) was added dropwise over 45 minutes to the reaction mixture, whichwas then stirred for a further 45 minutes at −45° C. TEA (1.0 g, 10.2mmol) was added dropwise to the mixture over 0.5 hours and stirred for afurther 15 minutes. The reaction mixture was left to warm to roomtemperature and diluted with H₂O (100 mL) and the phases separated. Theorganic phase was washed with 1N HCl (3×50 mL), water (3×50 mL), brine(3×50 mL) and dried over MgSO₄. Filtration and evaporation of excesssolvent afforded the product as an off-white glass (1.24 g, 2.3 mmol,92%); ¹H NMR (270 MHz, CDCl₃) δ−0.05 (s, 9H), 0.88-0.95 (m, 2H),2.06-2.23 (m, 4H), 3.46-3.64 (m, 2H), 3.75-4.02 (m, 7H), 4.11-4.27 (m,2H), 5.13 (s, 2H), 5.65 (d, 1H, J=9.71 Hz), 7.11 (s, 1H), 7.34-7.54 (m,5H); ¹³C NMR (67.8 MHz, CDCl₃) δ166.8, 157.2, 153.1, 150.5, 143.4,137.1, 129.2, 128.4, 128.3, 128.3, 128.1, 123.0, 106.2, 85.7, 75.0,64.7, 61.7, 59.8, 56.1, 46.4, 28.6, 23.0, 17.5, −1.5, −1.6.

(11S,11aS)-8,11-dihydroxy-7,9-dimethoxy-10-N-(2′-trimethylsilylethoxycarbonyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(129)

10% Pd/C catalyst (0.22 g) was added to a solution of the substrate 128(0.95 g, 2.1 mmol) in absolute EtOH (200 mL). The reaction mixture washydrogenated under pressure using a Parr hydrogenator at 55 psi H₂ for18 h. The reaction mixture was filtered through celite, and the celitewashed with hot EtOH, taking care not to allow the filtration pad to dryout. Removal of excess solvent afforded the product as a colourlessglass (0.84 g, 1.9 mmol, 92%); ¹H NMR (270 MHz, CDCl₃) δ0.07 (s, 9H),0.91-0.97 (m, 2H), 2.07-2.20 (m, 4H), 3.52-3.75 (m, 2H), 3.98-4.26 (m,9H), 5.65 (d, 1H, J=9.71 Hz), 6.26 (br s, 1H), 7.14 (s, 1H); ¹³C NMR(CDCl₃) δ167.0, 157.3, 146.8, 143.4, 141.3, 124.9, 123.5, 105.5, 105.2,85.8, 64.8, 64.6, 64.5, 61.2, 60.0, 56.4, 46.4, 28.9, 28.7, 23.1, 23.0,17.3, −1.3, −1.5, −1.7.

7,9-dimethoxy-8-Hydroxy-1,2,3,11a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepin-2-one(130)

A solution of TBAF in THF (4.3 mL of a 1N solution, 4.3 mmol) was addedto a rapidly stirred solution of 129 (0.37 g, 0.9 mmol) in THF (10 mL)and the reaction mixture heated to 35° C. for 2 h. The reaction mixturewas diluted with EtOAc (50 mL), dried over anhydrous MgSO₄, filtered andremoval of excess solvent by rotary evaporation under reduced pressureafforded the product as a brown oil (0.18 g, 0.7 mmol, 78%). H¹ NMR(CDCl₃) mixture of C11/C11′R/S carbinolamine methyl ethers δ7.08 (s,1H), 4.43 (d, 1H, J=8.79 Hz), 4.05-3.23 (m, 12H), 2.3-1.48 (m, 4H).

Examples 3(h) to (i)

Synthesis of 7-Phenyl PBDs (See FIG. 21)

Synthesis f the 7-Iodo-N10-Troc-PBD Intermediate (134, AG/91)

5-Iodo-2-(2′,2′,2′-trichloroethoxycarbonylamino)benzoic acid (132)

A solution of Troc-Cl (2.88 mL, 20.9 mmol) in dry dichloromethane (20mL) was added drop wise to a solution of 5-iodoanthranilic acid 131 (5g, 19 mmol) and pyridine (3.1 mL, 38 mmol) in dry dichloromethane (30mL) at 0° C. The solution was stirred for 5 hours at room temperatureand then washed with 1N HCl (2×25 mL), water (1×25 mL) and brine (1×25mL). The organic phase was dried over MgSO₄ and evaporated, residue wasrecrystallized from ethyl acetate to afford the title compound as ayellow solid (6.2 g, 75%): m.p. 248 C (ethyl acetate). ¹H NMR (CDCl₃,DMSO-d₆) δ4.83 (s, 2H); 7.78-7.82 (dd, J=9.2, J=2.2 Hz, 1H); 8.18 (d,J=9 Hz, 1H); 8.38 (d, J=2.2 Hz, 1H); 9.0-10.5 (bs, 1H); 11.04 (s, 1H).¹³C NMR (CDCl₃, DMSO-d₆) δ74.4, 84.6, 95.2, 117.7, 120.7, 140, 140.8,142.8, 151.5, 169. MS: m/e (relative intensity) 437 (M−1, 60), 289 (55),272 (37), 245 (100), 218 (27). HRMS Calculated for C₁₀H₇Cl₃INO₄:436.8485. Found: 436.8485.

N-(5-Iodo-(2′,2′,2′-trichloroethoxycarbonylamino)benzoyl)pyrrolidine-2-methanol(133)

Oxalyl chloride (0.88 mL, 10 mmol) was added to a suspension of 132 (4g, 9.1 mmol) in dry dichloromethane (50 mL), followed by 3-4 drops ofDMF as catalyst. The solution was stirred at room temperature for 12hours, and then used directly in the next step. The newly formed acidchloride was added drop wise, over 1 hour, to a solution of2S-(+)-pyrrolidinemethanol (1.01 g, 10 mmol) and triethylamine (3.16 mL,22.7 mmol) in dry dichloromethane (50 mL) at −20° C. The reactionmixture was allowed to stir for a further hour at −20° C. and was thenwashed with dilute HCl (1N, 2×50 mL), water (50 mL) and brine (50 mL),dried over MgSO₄ and evaporated. The crude product was subjected toflash column chromatography to afford the title compound as a paleyellow oil (3.8 g, 81%): ¹H NMR (CDCl₃, DMSO-d₆) δ1.77-2.28 (m, 4H);3.48 (bs, 2H); 3.7 (dd, J=11.4, J=6.2, 1H); 3.94 (d, J=11.4 Hz, 1H);4.40 (bs, 1H); 4.75 (d, J=12 Hz, 1H); 4.84 (d, J=12 Hz, 1H); 7.66-7.72(m, 2H); 7.85 (d, J=8.6 Hz, 1H); 8.91 (bs, 1H). ¹³C NMR (CDCl₃, DMSO-d₆)δ25.0, 28.1, 51.2, 60.7, 65.3, 74.5, 86.1, 95.1, 123.0, 128.0, 135.6,136.1, 139.8, 151.8, 168.4. IR (Nujol): cm⁻¹ 3415, 3215, 1745, 1605,1527, 1445, 1377, 1221, 1101, 1056, 822, 733. MS: m/e (relativeintensity) 522 (M^(+.), 3), 521 (M^(+.), 1), 520 (M^(+.), 3), 491 (3),490 (1), 489 (3), 372 (7), 341 (28), 272 (80), 245 (14), 216 (14), 83(15), 70 (100). HRMS Calculated for C₁₅H₁₆Cl₃IN₂O₄: 521.9193. Found:521.9125. [α]²⁵ _(D)=+123.4° (c=2.8, CHCl₃).

7-Iodo-10-N-(2′,2′,2′-trichloroethoxycarbonyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(134)

A solution of DMSO (1.79 mL, 25.67 mmol) in dry dichloromethane (35 mL)was slowly added (30 minutes) to a solution of oxalyl chloride (12.8mmol) in dry dichloromethane (41.4 mL) at −45° C. The mixture wasallowed to stir for 25 minutes and then treated with a solution of 133(4.78 g, 9.2 mmol), in dry dichloromethane (80 mL), keeping temperaturebelow −40° C. After further 60 minutes at −45° C., a solution oftriethylamine (5.1 mL) in of dichloromethane (25 mL) was added, and thereaction mixture allowed to warm to room temperature. The organic phasewas washed with water (180 mL), dilute HCl (1N, 2×100 mL) and brine (200mL). Removal of excess solvent afforded the crude product which waspurified by flash chromatography (ethyl acetate/petroleum ether 70/30)to give of a pale yellow oil (3.6 g, 76%): ¹H NMR (270 MHz, CDCl₃)δ2.02-2.15 (m, 4H); 3.37-3.60 (m, 2H); 3.70-3.77 (m, 1H); 4.19 (bs, 1H);4.28 (d, J=12 Hz, 1H); 5.17 (d, J=12 Hz, 1H); 5.66 (d, J=9.7 Hz, 1H);7.10 (d, J=8.3 Hz, 1H); 7.79 (dd, J=8.3, J=2.2 Hz, 1H); 8.10 (d, J=2.2Hz, 1H). ¹³C NMR (CDCl₃) δ23.0, 28.8, 46.5, 59.6, 75.1, 86.0, 93.2,94.8, 132.0, 133.6, 135.0, 137.9, 140.1, 154.1, 165.2. IR (Nujol): cm⁻¹3500-3000, 1716, 1619, 1458, 1376, 1312, 1075, 720. MS: m/e (relativeintensity) 520 (M^(+.), 62), 519 (22), 518 (62), 491 (15), 371 (19), 342(39), 272 (84), 216 (31), 119 (27), 70 (100). HRMS Calculated forC₁₅H₁₄Cl₃IN₂O₄: 519.9036. Found: 519.9037. [α]²⁵ _(D)=+137.4° (c=1.15,CHCl₃).

Example 3(h)

Synthesis of the 7-Phenyl-PBD (136, AG/129)

7-Phenyl-10-N-(2′,2′,2′-trichloroethoxycarbonyl)-1,2,3,10,11,11a-hexahydro-5-H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(135)

A suspension of 134 (0.5 g, 1.0 mmol), benzeneboronic acid (0.15 g, 1.22mmol), Pd(PPh₃)₄ and anhydrous Na₂CO₃ (0.16 g, 1.48 mmol) in distilledbenzene (20 mL), water (2 ml) and ethanol (2 mL) was heated at refluxovernight. The reaction mixture was diluted with ethyl acetate (20 mL)and washed with water (2×20 mL). The organic phase was dried over MgSO₄and evaporated to yield a crude yellow oil. Purification by flashchromatography (ethyl acetate/petroleum ether 30/70 to 70/30) furnishedthe title compound (0.43 g, 95%): ¹H NMR (270 MHz, CDCl₃) δ1.98-2.09 (m,2H); 2.12-2.15 (m, 2H); 3.51-3.62 (m, 2H); 3.7-3.79 (m, 1H); 4.28 (d,J=12.1 Hz, 1H); 4.73 (d, J=4.4 Hz, 1H); 5.18 (d, J=12.1 Hz, 1H);5.66-5.73 (dd, J=4.8, J=9.8 Hz, 1H); 7.33-7.48 (m, 4H); 7.61-7.70 (m,3H); 8.02 (d, J=2.2 Hz, 1H). ¹³C NMR (CDCl₃) δ22.9, 28.7, 46.4; 59.8;75.0; 77.3; 86.0; 94.9; 127.0; 127.3; 128.0; 128.9; 129.6; 130.8; 132.9;133.5; 139.2; 141.1; 154.4; 166.9. MS: m/e (relative intensity) 468(M^(+.), 10), 292 (25), 222 (100), 195 (10), 166 (35), 140 (10), 70(70). HRMS Calculated for C₂₁H₁₉C₁₃N₂O₄: 468.0411. Found: 468.0410.[α]²⁵ _(D)=+103.80 (c=0.42, CHCl₃).

(11aS)-7-Phenyl-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(136, AG/129)

Cd/Pb (0.47 g) couple was added portion wise to a vigorously stirredsolution of 135 (0.33 g, 0.7 mmol) in THF (5 mL) and of aq. ammoniumacetate (1M, 5 mL). The suspension was allowed to stir at roomtemperature for 2 hours, then poured into ethyl acetate (200 mL), driedwith MgSO₄ and filtered. The filtrate was evaporated and the residuepurified by flash column chromatography (ethyl acetate) to afford thetitle compound as colourless oil (0.19 g, 98%): ¹H NMR (270 MHz, CDCl₃)δ2.0-2.12 (m, 2H); 2.29-2.37 (m, 2H); 3.53-3.63 (m, 1H); 3.76-3.92 (m,2H); 7.36-7.79 (m, 8H); 8.28 (d, J=2.2 Hz, 1H). ¹³C NMR (67.8 MHz,CDCl₃) δ24.4; 29.8; 46.9; 53.8; 126.9; 127.3; 127.7; 128.0; 128.2;128.8; 128.9; 129.1; 130.1; 130.5; 139.5; 145.0; 164.5; 165.1. IR(Nujol): cm⁻¹ 3000-2800, 1620, 1455, 1377, 1239, 1239, 1014, 990,761,728, 697. HRMS Calculated for C₁₈H₁₆N₂O: 276.1261. Found: 276.1262.[α]²⁵ _(D)=+131.4° (c=0.19, CHCl₃).

Example 3(i)

Synthesis of the 7-(4′-Methoxyphenyl)-PBD (138, AG/135)

(11S,11aS)-7-(4′-Methoxy)phenyl-11-hydroxy-10-N-(2″,2″,2″-trichloroethoxycarbonyl)-1,2,3,10,11a-hexahydro-5H-pyrrolo]2,1-c]-[1,4]benzodiazepin-5-one(137)

134 (0.5 g, 1.0 mmol), 4-methoxybenzeneboronic acid (0.19 g, 1.2 mmol),Pd(PPh₃)₄ (15 mg) and anhydrous Na₂CO₃ (0.16 g, 1.48 mmol) were heatedat reflux, over night, in a mixture of distilled benzene (20 mL),ethanol (2 mL) and water (2 mL). The reaction mixture was diluted withethyl acetate (20 mL) and washed with water (2×20 mL). The organic phasewas dried over MgSO₄ and evaporated to yield a crude yellow oil.Purification by flash chromatography (ethyl acetate/petroleum ether50/50) afforded the pure compound (0.34 g, 71%): ¹H NMR (CDCl₃)δ1.96-2.16 (m, 4H); 3.54-3.63 (m, 2H); 3.71-3.79 (m, 1H); 3.85 (s, 3H);4.18 (d, J=4.8 Hz, 1H); 4.29 (d, J=12.1 Hz, 1H); 5.20 (d, J=12.1 Hz,1H); 5.66-5.72 (dd, J=4.5, J=9.8 Hz, 1H); 6.97 (d, J=8.8 Hz, 2H); 7.37(d, J=8.2 Hz, 1H); 7.57 (d, J=8.8 Hz, 2H); 7.64 (dd, J=2.4, J=8.2 Hz,1H); 7.97 (d, J=2 Hz, 1H). ¹³C NMR (67.8 MHz, CDCl₃) δ23.0; 28.7; 46.4;55.4; 59.6; 75.1; 86.1; 94.9; 114.3; 126.8; 129.1; 130.6; 131.7; 132.0;132.2; 132.3; 133.5; 140.7; 154.5; 159.6; 166.9. IR (Nujol): cm⁻¹3000-2800, 1740, 1620, 1462, 1378, 1247, 1082, 816, 721. MS: m/e(relative intensity) 498 (M^(+.), 15), 350 (20), 321 (15), 252 (100),196 (22), 182 (5), 126 (7), 70 (28). HRMS Calculated for C₂₂H₂₁Cl₃N₂O₅:498.0515. Found: 498.0513. [α]²⁵ _(D)=+149.4° (0.25, CHCl₃)

(11aS)-7-(4′-Methoxyphenyl)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c]-[1,4]benzodiazepin-5-one(138, AG/135)

Cd/Pb couple (0.51 g) was added portion wise to a, vigorously stirred,solution of 137 (0.34 g, 0.76 mmol) in THF (5 mL) and aq. ammoniumacetate (1M, 5 mL). The suspension was allowed to stir at roomtemperature for 2 hours, then poured into ethyl acetate (200 mL), driedover MgSO₄ and filtered. The organic solution was evaporated and theresidue purified by flash column chromatography (ethyl acetate), toafford the title compound as colourless oil (0.1 g, 70%): ¹H NMR (CDCl₃,DMSO-d₆) δ2.1 (m, 2H); 2.3-2.4 (m, 2H); 3.5-3.62 (m, 1H); 3.85 (m, 5H);7.0 (d, J=8.8 Hz, 2H); 7.36 (d, J=8.3 Hz, 2H); 7.6 (d, J=8.8 Hz, 2H);7.72 (dd, J=2.2, J=8.2 Hz 1H); 7.8 (d, J=4.4 Hz, 1H,); 8.2 (d, J=2.2 Hz,1H). ¹³C NMR (270 MHz, CDCl₃, DMSO-d₆) δ24.1; 29.5; 46.7; 53.6; 55.3;77.3; 114.1; 114.3; 127.4; 127.6; 127.8; 128.0; 129.3; 131.9; 138.7;144.3; 159.4; 164.2; 164.8. IR (Nujol): cm⁻¹ 3000-2800, 1662, 1607,1491, 1454, 1245, 1069, 823, 759. MS: m/e (relative intensity) 306(M^(+.), 100), 277 (15), 237 (10), 182 (12), 153 (10), 132 (5), 70 (10).HRMS Calculated for C₁₉H₁₈N₂O₂: 306.1367. Found: 306.1365. [α]²⁵_(D)=+773.1° (c=0.11, CH₃OH).

Example 3(j)

Synthesis of the 7-(3′-Nitrophenyl)-PBD (140, AG/150)

(11S,11aS)-7-(3′-Nitro)phenyl-11-hydroxy-10-N-(2″,2″,2″-trichloroethoxycarbonyl)-1,2,3,10,11a-hexahydro-5H-pyrrolo[2,1-c]-[1,4]benzodiazepin-5-one(139)

134 (0.5 g, 1.0 mmol), 3-nitrobenzeneboronic acid (0.2 g, 1.2 mmol),Pd(PPh₃)₄ (25 mg) and anhydrous Na₂CO₃ (0.16 g, 1.48 mmol) were heatedat reflux, over night, in a mixture of distilled benzene (20 mL),ethanol (2 mL) and water (2 mL). The reaction mixture was diluted withethyl acetate (20 ml) and washed with water (2×20 ml). The organic phasewas dried over MgSO₄ and evaporated to yield a crude yellow oil.Purification by flash chromatography (ethyl acetate/petroleum ether50/50) afforded the pure compound (0.45 g, 90%): ¹H NMR (270 MHz, CDCl₃)δ2.0-2.2 (m, 4H); 3.6 (m, 2H); 3.76 (m, 1H); 4.31 (d, J=12 Hz, 1H); 5.19(d, J=12 Hz, 1H); 5.76 (d, J=10 Hz, 1H); 7.5-8.5 (m, 8H). ¹³C NMR (68.7MHz, CDCl₃) δ22.9, 28.7, 46.4, 59.7, 75.0, 86.0, 94.8, 121.7, 122.6,127.5, 129.4, 129.9, 131.2, 132.0, 132.8, 133.9, 138.3, 140.7, 148.6,154.1, 166.3. IR (Nujol): cm⁻¹ 3000-2800, 1721, 1626, 1530, 1455, 1349,1062, 821, 759. MS: m/e (relative intensity) 513 (M^(+.)), 336 (55), 321(100), 292 (15), 267 (54), 221 (16), 197 (18), 164 (15), 70 (22). HRMSCalculated for C₂₁H₁₈C₁₃N₃O₆: 515.0233. Found: 515.0235. [α]²⁵_(D)=+129.6° (c=0.1, CH₃OH)

(11aS)-7-(3′-Nitrophenyl)-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(140, AG-150)

A solution of TBAF in THF (1M solution, 7.6 mL, 7.6 mmol) was added to asolution 139 (0.39 g, 0.8 mmol) in of THF (20 mL) and the reactionmixture allowed to stir for 2 hours at room temperature. The solutionwas diluted with ethyl acetate (50 mL) and washed with water (3×50 mL)to remove excess TBAF. The organic phase was dried over MgSO₄ andevaporated to dryness. The residue was purified by flash columnchromatography (CHCl₃), to afford the title compound as a colourless oil(0.15 g, 63%): ¹H NMR (270 MHz, CDCl₃) δ1.8-2.2 (m, 3H); 3.5-4.0 (m,3H); 7.3-8.5 (m, 7H). IR (Nujol): cm⁻¹ 3000-2850, 1624, 1527, 1466,1349, 1244, 757, 740. MS: m/e (relative intensity) 321 (M^(+.), 100),292 (8), 265 (5), 224 (5), 197 (7), 151 (5), 70 (5). HRMS Calculated forC₁₈H₁₅N₃O₃: 321.1115. Found: 321.1113. [α]²⁵ _(D)=+129.6° (c=0.1,CH₃OH).

Example 3(k)

8-Benzyloxy-7,9-dimethoxy-1,2,3,11a-tetrahydropyrrolo]2,1-c][1,4]benzodiazepin-5-one(143, DRH-105) (see FIG. 22)

N-(4-Benzyloxy-3,5-dimethoxy-2-[trichloroethyloxycarbonylamino]benzoyl)pyrrolidine-2-methanol(141)

A solution of 2,2,2-trichloroethyl chloroformate (1.08 g, 4.8 mmol) indistilled dichloromethane (10 mL) was added dropwise over 0.5 hours to asolution of anhydrous pyridine (0.80 g, 10.1 mmol) and 126 (Example3(g))(1.95 g, 5.1 mmol) in distilled dichloromethane (20 mL) at 0° C.After 1 hour the reaction mixture was diluted with anhydrous DCM (100mL) and washed with 1N HCl (2×100 mL), H₂O (100 mL), brine (100 mL) anddried over anhydrous MgSO₄. Evaporation of the solvent yielded a brownoil which was purified by flash column chromatography (silica gel,EtOAc) to afford the product as a yellow glass (2.65 g, 4.7 mmol, 94%);¹H NMR (270 MHz, CDCl₃) δ1.6-2.2 (m, 4H), 3.3-3.6 (m, 2H), 3.6-3.9 (m,2H), 3.8 (s, 3H), 3.9 (s, 3H), 4.2-4.3 (m, 1H), 4.8 (s, 2H), 5.1 (s,2H), 6.6 (s, 1H), 7.2 (br s, 1H), 7.3-7.5 (m, 5H); ¹³C NMR (67.8 MHZ,CDCl₃) δ171.5, 153.1, 142.0, 137.023, 128.3, 128.3, 128.2, 120.1, 105.3,95.4, 75.3, 74.6, 66.5, 61.4, 61.3, 56.3, 50.7, 28.7, 24.6.

(11S,11aS)-8-benzyloxy-7,9-dimethoxy-11-hydroxy-10-N-(2′,2′,2′-trichloroethoxylcarbonyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(142)

Anhydrous DMSO (0.97 g, 12.5 mmol) in dry DCM (10 mL) was added dropwiseover 30 minutes to a stirred solution of oxalyl chloride (3.08 mL of a2N solution in DCM, 6.2 mmol) in dry DCM (10 mL) under a nitrogenatmosphere at −45° C. After stirring for 15 minutes, the substrate (2.46g, 4.38 mmol) in dry DCM (25 mL) was added dropwise over 45 minutes tothe reaction mixture, which was then stirred for a further 45 minutes at−45° C. TEA (1.77 g; 17.5 mmol) was added dropwise to the mixture over0.5 hours and stirred for a further 15 minutes. The reaction mixture wasleft to warm to room temperature, diluted with H₂O (100 mL)and thephases allowed to separate. The organic phase was washed with 1N HCl(2×50 mL), water (2×50 mL), brine (2×50 mL) and dried over MgSO₄. Thesolvent was evaporated to afford the product as an off-white glass (3.92g, 11.7 mmol; 97%); ¹H NMR (270 MHz, CDCl₃) δ2.01-2.17 (m, 4H),3.44-3.77 (m, 2H), 3.87-3.90 (m, 1H), 3.88 (s, 3H), 3.91 (s, 3H), 4.68(dd, 2H), 5.01 (s, 2H), 5.62 (d, 1H), 7.08 (s, 1H), 7.27-7.48 (m, 5H);¹³C NMR (67.8 MHz, CDCl₃) δ166.7, 155.2, 153.6, 150.5, 143.6, 137.1,129.8, 129.3, 128.4, 128.3, 128.2, 128.1, 121.8, 106.5, 106.3, 94.7,86.2, 85.9, 75.6, 75.4, 75.2, 75.0, 61.8, 61.5, 60.2, 59.870, 56.1,56.0, 46.5, 46.3, 45.8, 28.7, 28.6, 23.0.

8-Benzyloxy-7,9-dimethoxy-1,2,3,11a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepin-5-one(143)

10% Cd/Pb couple (1.2 g; 10 mmol Cd) was added to a rapidly stirringsolution of 142 (1.08 g; 1.9 mmol) in a mixture of THF (15 mL) and 1NNH₄OAc (15 mL). After 3.5 h, TLC revealed that reaction was stillincomplete and more 10% Cd/Pb couple (500 mg) was added. After a further1 hour the reaction mixture was diluted with EtOAc (150 mL). Thesolution was dried over anhydrous MgSO₄ and the solids were filtered andrinsed with EtOAc (50 mL). Removal of excess solvent yielded the productas a yellow glass (0.48 g, 1.3 mmol, 68%). H¹ NMR (270 MHz, CDCl₃) δ7.73(d, 1H, J=4.4 Hz), 7.36 (s, 2H), 7.31 (s, 2H), 7.11 (s, 1H), 7.08 (s,1H), 5.12 (br s, 2H), 3.98-3.42 (m, 9H), 2.38-2.29 (m, 2H), 2.23-1.83(m, 2H).

Example 3(1)

Synthesis of the C8-NH₂ PBD (151, AG/149) (see FIG. 23)

4-Nitro-2-(2′,2′,2′-trichloroethoxycarbonylamino)benzoic acid (145)

A solution of 2,2,2-trichloroethylchloroformate (Troc-Cl) (1.66 mL, 12.1mmol) in dry dichloromethane (25 mL) was added drop wise to a solutionof 4-nitroanthranilic acid 144 (2 g, 11 mmol) and pyridine (1.78 mL, 22mmol) in dichloromethane (25 ml) at 0° C. The solution was allowed tostir at 25° C. for 5 hours. The reaction mixture was washed with diluteHCl (1N, 2×50 mL), water (1×50 mL), brine (1×25 mL) and dried overMgSO₄. Removal of excess solvent by rotary evaporation under reducedpressure afforded the crude product which was used in the subsequentreaction without further purification.

N-[4-nitro-(2′,2′,2′-trichloroethoxycarbonylamino)benzoyl]pyrrolidine-2-methanol(146)

Oxalyl chloride (1 mL, 12.1 mmol) and a catalytic amount of dry DMF wereadded to a suspension of the crude product from the previous reaction inof dry dichloromethane (50 mL) and the reaction mixture was allowed tostir at room temperature for 12 hours. The newly formed acid chloridewas added drop wise, over 1 hour, to a solution of2S-(+)-pyrrolidinemethanol (1.22 g, 12.1 mmol) and triethylamine (3.8mL, 27.5 mmol) in dichloromethane (50 mL) at −20° C. (CCl₄-dry ice). Thereaction mixture was stirred for a further hour at −20° C. and was thenallowed to warm to room temperature. The reaction mixture was washedwith dilute HCl (1N, 2×50 mL), water (50 mL) and brine (50 mL), driedover MgSO₄ and evaporated under reduced pressure. The residue waspurified by flash chromatography (EtOAc/petroleum ether 50/50), removalof excess eluent afforded of a yellow oil (1.34 g, 30%, over two steps):¹H NMR (270 MHz, CDCl₃) δ1.7-2.3 (m, 4H); 3.45 (m, 2H); 3.71 (dd, J=5.5,J=11, 1H); 4.06 (m, 2H); 4.43 (bs, 1H); 4.85 (d, J=13, 1H); 4.89 (d,J=13 Hz, 1H); 7.56 (d, J=8.4 Hz, 1H); 7.96 (dd, J=2.2, J=8.4 Hz, 1H);8.94 (d, J=2.2 Hz, 1H); 9.2 (bs, 1H). ¹³C NMR (67.8 MHz, CDCl₃) δ24.9;27.9; 50.8; 60.5; 64.3; 74.6; 94.9; 115.9; 117.9; 128.6; 130.5; 136.9;149.0; 151.8; 167.7. MS: m/e (relative intensity) 441 ([M+1], 1), 291(10), 260 (12), 191 (30), 164 (15), 154 (8), 113 (20), 77 (20), 70(100). HRMS Calculated for C₁₅H₁₆C₁₃N₃O₆: 439.0104. Found: 439.0105.[α]²⁵ _(D)=−110.6° (c=0.13, CHCl₃).

N-[4-amino(2′,2′,2′-trichloroethoxycarbonylamino)benzoyl]pyrrolidine-2-methanol(147)

A solution of 146 (1 g, 2.3 mmol) and SnCl₂2H₂O (2.56 g, 11.4 mmol) inmethanol (20 mL) was heated at reflux for 6 hours (the reaction wasmonitored by TLC (3% methanol, ethyl acetate). The reaction mixture wasreduced to ⅓ of it's original volume and the pH adjusted to 8-9 withsatd. aqueous NaHCO₃. Ethyl acetate (100 mL) was added and the mixturewas vigorously stirred for 12 hours, then filtered through Celite toremove tin salts. The organic phase was dried over MgSO₄ and evaporatedto afford the product as a yellow oil (0.94 g, 97%) which was used inthe next reaction without further purification: ¹H NMR (270 MHz, CDCl₃)δ1.6-1.8 (m, 2H); 1.9 (m, 1H); 2.17 (m, 1H); 3.48-3.58 (m, 1H);3.62-3.72 (m, 2H); 3.84 (m, 1H); 4.44 (m, 1H); 4.77 (d, J=12.1 Hz , 1H);4.83 (d, J=12.1 Hz, 1H); 6.32 (dd, J=2.2, J=8.43 Hz, 1H); 7.18 (d,J=8.43 Hz, 1H); 7.52 (d, J=2.2 Hz, 1H); 9.62 (bs, 1H). ¹³C NMR (67.8MHz, CDCl₃) δ21.1; 25.2; 28.2; 51.9; 60.9; 66.5; 74.3; 95.3; 105.5;108.3; 112.6; 130.1; 138.9; 149.7; 151.8; 171.5. IR (Nujol): cm⁻¹ 3346,3000-2800, 1738, 1620, 1463, 1196, 1046, 963, 820 760. MS: m/e (relativeintensity) 409 ([M−1], 15), 309 (20), 179 (25), 161 (100), 134 (8), 113(25), 77 (35), 70 (85). HRMS Calculated for C₁₅H₁₈Cl₁₃N₃O₄: 409.0362.Found: 409.0363. [α]²⁵ _(D)=−60.1° (c=0.3, CHCl₃).

N-[4-(Fmoc)amino(2′,2′,2′-trichloroethoxycarbonylamino)benzoyl]pyrrolidine-2-methanol (148)

An aqueous solution of NaHCO₃ (0.6 g, 5.67=mol, in 20 mL of H₂O) wasadded to a solution of 147 (0.94 g, 2.3 mmol) in THF (20 mL). Thereaction mixture was cooled to 0° C. and Fmoc-Cl (0.65 g, 2.5 mmol) wasadded in small portions. After addition the reaction mixture was allowedto stir for 2 hours at room temperature. (TLC: ethyl acetate/petroleumether 50/50). The reaction mixture was acidified with dilute HCl (1N)and extracted with ethyl acetate (2×20 mL). The organic phase was dried(MgSO₄) and evaporated and the resulting yellow oil thus obtained waspurified by flash chromatography to afford the product (1.03 g, 72%): ¹HNMR (270 MHz, CDCl₃) δ1.68 (m, 2H); 1.84 (m, 1H); 2.11 (m, 1H); 3.48 (m,2H); 3.71 (m, 1H); 3.87 (m, 1H); 4.19 (t, J=6.8 Hz, 1H); 4.40 (m, 2H);4.45 (d, J=6.78 Hz, 2H); 4.73 (d, J=12.1, 1H); 4.78 (d, J=12.1 Hz, 1H);7.2-7.8 (m, 11H); 8.04 (bs, 1H). ¹³C NMR (67.8 MHz, CDCl₃) δ25.1; 28.1;46.8; 51.6; 60.8; 65.7; 67.1; 74.3; 95.2; 109.9; 112.3; 118.3; 120.0;124.9; 127.1; 127.8; 129.3; 137.5; 140.9; 141.2; 143.6; 151.8; 153.2;170.3. IR (Nujol): cm⁻¹ 3301, 3000-2800, 1738, 1599, 1525, 1451, 1224,1056, 985, 758, 740, 667. MS: m/e (relative intensity) 632 (M^(+.)), 409(15), 309 (20), 179 (25), 161 (100), 134 (8), 113 (25), 77 (35), 70(85). [α]²⁵ _(D)=−70.3° (c=0.25, CHCl₃).

(11S,11aS)-8-(Fmoc)amino-11-hydroxy-10-N-(2′,2′,2′-trichloroethoxycarbonyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(149)

A solution of DMSO (0.31 ml, 4.4 mmol) in of dry dichloromethane (10 mL)was slowly added (over 30 minutes) to a solution of oxalyl chloride (2.2mmol) in dry dichloromethane (11.1 mL) at −45° C. The mixture wasallowed to stir for 15 minutes followed by the addition of a solution of148 (1 g, 1.58 mmol) in of dry dichloromethane (15 ml), keeping thetemperature below −40° C. After further 60 minutes at −45° C., asolution of triethylamine (0.88 ml 6.32 mmol) in dichloromethane (6 mL)was added and the reaction mixture allowed to warm to room temperature.The reaction mixture was washed with water (50 mL), dilute HCl (1N, 50mL) and brine (50 mL). Evaporation of solvent afforded the crude productwhich was purified by flash chromatography (ethyl acetate/petroleumether 50/50). Removal of excess eluent furnished the product as a paleyellow oil (0.81 g, 82%): ¹H NMR (CDCl₃) δ1.96-2.16 (m, 4H); 3.47-3.56(m, 3H); 3.6 (m, 1H); 4.1-4.28 (m, 3H); 4.46 (d, J=6.15 Hz, 2H); 5.01(d, J=12.1 Hz, 1H); 5.64 (d, J=12.1 Hz 1H); 7.22-7.76 (m, 11H). ¹³C NMR(67.8 MHz, CDCl₃) δ22.9; 28.7; 46.4; 46.9; 59.9; 67.0; 75.1; 86.0; 94.8;117.7; 119.6; 120.1; 124.9; 127.9; 129.8; 134.9; 140.8; 141.3; 143.5;153.0; 154.1; 166.7. IR (Nujol): cm⁻¹ 3282, 3000-2800, 1713, 1610, 1533,1451, 1220, 1058, 908, 735, 647 MS: m/e (relative intensity) 631 ([M+2],1), 196 (5), 178 (100), 152 (5), 89 (7), 70 (10). HRMS Calculated forC₃₀H₂₆Cl₃N₃O₆: 629.0887. Found: 629.0887. [α]²⁵ _(D)=+58.7° (c=0.5,CHCl₃).

(11S,11aS)-8-amino-11-hydroxy-10-N-(2′,2′,2′-trichloroethoxycarbonyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(150)

The protected carbinolamine 149 (0.8 g, 1.3 mmol) was added to a 5%solution of piperidine in CH₃CN (12 mL, 5 eq. of piperidine). Themixture was allowed to stir for 12 hours, extracted with water (2×50 mL)and the organic phase was evaporated under reduced pressure to yield apale yellow oil (0.24 g, 50%): ¹H NMR (270 MHz, CDCl₃) δ1.9-2.2 (m, 4H);3.45-3.7 (m, 3H); 4.26 (d, J=12.1 Hz, 1H); 4.55 (m, 3H); 5.18 (d, J=12.1Hz, 1H); 5.61 (d, J=10.3 Hz, 1H); 6.61 (s, 1H); 6.69 (d, J=7.3 Hz, 1H);7.56 (d, J=8.2 Hz, 1H). ¹³C NMR (67.8 MHz, CDCl₃) δ23.0; 28.7; 46.3;59.8; 74.9; 95.1; 114.8; 116.5; 130.4; 135.3; 154.4; 167.3. IR (Nujol):cm⁻¹ 3340, 3224, 3000-2800, 1714, 1602, 1460, 1311, 1208, 1141, 1061,826, 759, 665. MS: m/e (relative intensity) 407 (M^(+.) , 40), 381 (5),340 (10), 309 (25), 161 (100), 134 (15), 105 (15), 70 (80). HRMSCalculated for C₁₅H₁₆C₁₃N₃O₄. 407.0206. Found: 407.0206. [α]²⁵_(D)=+47.8° (c=0.5, CHCl₃).

Synthesis of(11aS)-8-amino-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(151)

Cd/Pb couple (5 eq, 0.34 g) was added portion wise to a vigorouslystirred solution of 150 (0.2 g, 0.5 mmol) in THF (10 mL) and aqueousammonium acetate (10 mL). Stirring was allowed to continue for a further2 hours at room temperature and the reaction mixture was poured intoethyl acetate (100 mL). The organic phase was dried over MgSO₄, filteredand evaporated to yield the crude product which was subjected to flashchromatography (silica gel, 5% MeOH, 95% CHCl₃). Removal of excesseluent afforded the product as a white solid (26 mg, 53% yield): ¹H NMR(270 MHz, CDCl₃, CD₃OD) δ1.6-2.2 (m, 4H); 3.2-3.4 (m, 2H); 3.5 (m, 1H);5.0 (m, 2H); 6.05 (m, 1H); 6.25 (m, 1H); 7.43 (m, 1H), 7.75 (m, 1H). IR(Nujol): cm⁻¹ 3304, 3000-2800, 1613, 1457, 1377, 1244, 1202, 1122, 1072,825, 759, 721. MS: m/e (relative intensity) 215 (M^(+.), 100), 186 (15),178 (10), 146 (10), 119 (25), 91 (15),70 (30), 65 (5). HRMS Calculatedfor C₁₂H₁₃N₃O: 215.1058. Found: 215.1059. [α]²⁵ _(D)=+163.3° (c=0.2,CHCl₃).

Example 3(m)

Synthesis of(11aS)-8-methyl-7,9-dimethoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(194) (see FIG. 24)

Methyl 4-methyl-3,5-dimethoxybenzoate (187)

Concentrated sulphuric acid (1 mL), was added dropwise to a solution of4-methyl-3,5-dimethoxybenzoic acid (186) (5.01 g, 25.56 mmol) inrefluxing methanol (20 mL). The reaction mixture was heated at refluxfor a further 5 hours and then cooled to room temperature andconcentrated to a third of its original volume. The concentrate waspoured onto crushed ice (c. 150 mL) and allowed to stand for 30 minutes.The aqueous mixture was extracted with ethyl acetate (3×100 mL) and thecombined organic phase washed with distilled water (3×100 mL), brine(2×100 mL) and dried over anhydrous MgSO₄. Removal of excess solventunder reduced pressure afforded the product as a beige solid (187)(4.865 g, 23.17 mmol, 91%); ¹H NMR (270 MHz, CDCl₃) δ7.21 (s, 2H), 3.91(s, 3H), 3.86 (s, 6H), 2.13 (s, 3H); ¹³C NMR (67.8 MHz, CDCl₃) δ171.57,167.28, 158.16, 158.10, 128.23, 120.39, 105.20, 104.70, 55.85, 52.13,8.77, 8.66.

Methyl 2-nitro-4-methyl-3,5-dimethoxybenzoate (188)

Finely ground copper nitrate (Cu(NO₂)₃, 5.37 g, 28.57 mmol) was addedportionwise to a vigorously stirred solution of 187 (4.8 g, 22.86 mmol)in acetic anhydride (30 mL), whilst keeping the reaction temperaturebelow 40° C. The reaction mixture was stirred for 2 hours and thenpoured onto crushed ice (800 mL). The aqueous mixture was left to stirfor 1 hour and the product collected by filtration to afford a yellowsolid (188) (4.88 g, 18.945 mmol); ¹H NMR (270 MHz, CDCl₃) δ7.20 (br s,1H), 3.92 (s, 3H), 3.89 (s, 3H), 3.84 (s, 3H), 2.21 (s, 3H); ¹³C NMR(67.8 MHz, CDCl₃) δ163.70, 159.01, 150.89, 140.08, 127.06, 121.54,106.98, 62.88, 56.21, 52.98, 9.82.

2-Nitro-4-methyl-3,5-dimethoxybenzoic acid (189)

Potassium hydroxide (3.71 g, 66.31 mmol) was added to a stirred solutionof 188 (4.83 g, 18.95 mmol) in anhydrous methanol (80 mL) and thereaction mixture heated at reflux for 3 h. The reaction mixture wasallowed to cool and acidified to pH2 with 1 N HCl and the solidprecipitate was filtered and washed with water (50 mL) and left to airdry to afford the product as a yellow-beige solid (189) (3.69 g, 15.31mmol, 81%); ¹H NMR (270 MHz, CDCl₃) δ13.88 (br s, 1H), 7.23 (s, 1H),3.90 (s, 3H), 3.75 (s, 3H), 2.14 (s, 3H); ¹³C NMR (67.8 MHz, CDCl₃)δ164.09, 158.65, 150.09, 139.38, 125.70, 122.50, 107.24, 62.78, 56.34,9.62.

N-(4-Methyl-3,5-dimethoxy-2-nitrobenzoyl)pyrrolidine-2-methanol (190)

A catalytic amount of DMF (2 drops) was added to a stirred solution of189 (3.96 g, 15.32 mmol) and oxalyl chloride (2.14 g, 16.85 mmol) in dryCH₂Cl₂ (50 mL) under a nitrogen atmosphere. The reaction mixture wasallowed to stir overnight and the resulting acid chloride used directlyin the next stage of the procedure.4-methyl-3,5-dimethoxy-2-nitro-benzoyl chloride in anhydrous DCM (50 mL)was added dropwise over 0.5 hours to a stirring solution of pyrrolidinemethanol (1.55 g, 15.32 mmol, 1.1 eq) and TEA (3.87 g, 38.3 mmol, 2.5eq) in anhydrous DCM (50 mL) at 0° C. under a nitrogen atmosphere andthe reaction mixture was allowed to stir overnight at room temperature.The reaction mixture was washed with 1 N HCl (2×100 mL), and the organiclayer was washed with distilled H₂O (2×100 mL), brine (2×100 mL) anddried over anhydrous MgSO₄. Evaporation of excess solvent yielded ayellow glass (190) (2.13 g, 6.56 mmol, 43% -2 steps); ¹H NMR (270 MHz,CDCl₃) δ6.61 (s, 1H), 4.30-4.28 (m, 1H), 3.91 (s, 3H), 3.89 (s, 3H),3.83-3.68 (m, 2H), 3.46-3.26 (m, 2H), 2.19 (s, 3H), 1.94-1.68 (m, 4H);¹³C NMR (67.8 MHz, CDCl₃) δ167.85, 161.15, 152.62, 135.70, 132.32,123.17, 103.70, 65.87, 62.61, 61.37, 56.36, 50.20, 28.41, 24.50, 9.34.

N-(2-Amino-4-methyl-3,5-dimethoxybenzoyl) pyrrolidine-2-methanol (191)

Hydrazine hydrate (1.26 g, 39.37 mmol) was added dropwise to a solutionof 190 (2.13 g, 6.56 mmol) in methanol (50 mL) gently refluxing overRaney nickel (1 g, slurry). The resulting vigorous evolution of hydrogengas subsided after approximately 10 minutes and the reaction was deemedto be complete by TLC after 2 h. The reaction mixture was filteredthrough celite and the solvent evaporated. Distilled water (100 mL) wasadded to the residue, and the aqueous mixture was extracted with EtOAc(3×100 mL) and the combined organic phase washed with H₂O (3×100 mL) andbrine (3×100 mL) and dried over anhydrous MgSO₄. Evaporation of thesolvent afforded the product as a brown glass (191) (1.91 g, 6.50 mmol)which was protected directly as the troc-carbamate.

N-(4-Methyl-3,5-dimethoxy-2-[trichloroethyloxycarbonylamino]-benzoyl)pyrrolidine-2-methanol(192)

A solution of 2,2,2-trichloroethyl chloroformate (1.38 g, 6.5 mmol) indistilled DCM (25 mL) was added dropwise over 0.5 hours to a solution ofanhydrous pyridine (1.03 g, 13 mmol) and 191 (1.91 g, 6.5 mmol) indistilled DCM (25 mL) at 0° C. After 6 hours at room temperature, thereaction mixture was diluted with anhydrous DCM (100 mL) and washed with1 N HCl (2×100 mL), H₂O (100 mL), brine (100 mL) and dried overanhydrous MgSO₄. Evaporation of the solvent yielded a brown oil whichwas purified by flash column chromatography (silica gel, EtOAc) toafford the product (192) as a yellow glass (2.13 g, 4.53 mmol, 70%); ¹HNMR (270 MHz, CDCl₃) δ7.59 (br s, 1H), 6.56 (s, 1H), 4.78 (br s, 2H),4.25-4.23 (m, 1H), 3.82-3.79 (m, 3H+1H), 3.69-3.63 (m, 3H+1H), 3.52 (m,1H), 3.42-3.33 (m, 1H), 2.13-2.06 (m, 3H+1H), 1.88-1.64 (m, 3H); ¹³C NMR(67.8 MHZ, CDCl₃) δ169.95, 156.82, 156.67, 154.02, 153.34, 131.92,121.74, 119.33, 103.94, 95.43, 74.42, 66.04, 61.01, 60.60, 60.27, 55.73,50.46, 28.53, 24.47, 9.13.

(11S,11aS)-8-Methyl-7,9-dimethoxy-11-hydroxy-10-N-(2′,2′,2′-tri-chloroethoxylcarbonyl)-1,2,3,10,11,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(193)

Anhydrous DMSO (1.006 g, 12.87 mmol) in dry DCM (10 mL) was addeddropwise over 5 minutes to a stirred solution of oxalyl chloride (3.19mL of a 2 N solution in DCM, 6.373 mmol) under a nitrogen atmosphere at−50° C. After stirring for 5 minutes, a solution of 192 (2.13 g, 4.53mmol) in dry DCM (10 mL) was added dropwise over 45 minutes to thereaction mixture, which was then stirred for a further 45 minutes at−50°=0 C. TEA (1.83 g; 18.13 mmol) was added dropwise to the mixtureover 0.5 hours and stirred for a further 15 minutes. The reactionmixture was left to warm to room temperature, diluted with H₂O (100mL)and the phases allowed to separate. The organic phase was washed with1 N HCl (2×50 mL), water (2×50 mL), brine (2×50 mL) and dried overMgSO₄. The solvent was evaporated to afford the product (193) as anoff-white glass (1.84 g, 3.93 mmol; 87%). ¹H NMR (270 MHz, CDCl₃) δ7.05(s, 1H) (minor rotamer 1:4, visible at d 7.06), 5.58 (dd, J=3.84 Hz,J=9.89, 1H) (minor rotamer 1:4, visible at d 5.68, J=4.21 Hz, J=9.53),4.71 (d, J=11.72 Hz, 1H), 4.56 (d, J=11.73 Hz, 1H), 4.11 (d, J=3.85 Hz,1H), 3.88 (s, 3H), 3.76 (s, 3H), 3.79-3.47 (m, 2H), 2.21-1.99 (m, 4H),2.16 (s, 3H); ¹³C NMR (67.8 MHz, CDCl₃) rotamers δ166.82, 158.31,157.92, 155.65, 155.43, 131.52, 124.17, 123.99, 121.62, 105.90, 105.64,105.38, 94.53, 86.17, 85.93, 75.80, 75.23, 61.77, 61.65, 59.98, 59.58,59.40, 55.88, 55.79, 46.56, 46.38, 28.70, 28.62, 22.94, 10.14, 9.75.

8-Methyl-7,9-dimethoxy-1,2,3,11a-tetrahydropyrrolo[2,1-c][1,4]benzodiazepin-5-one(194)

10% Cd/Pb couple (1.34 g; 10.7 mmol Cd) was added to a rapidly stirringsolution of 193 (1 g; 2.14 mmol) in a mixture of THF (15 mL) and 1 NNH₄OAc (15 mL). After 3.5 hours the reaction was diluted with EtOAc (150mL). The solution was dried over anhydrous MgSO₄ and the solids werefiltered and rinsed with EtOAc (50 mL). Removal of excess solventyielded the product (194) as a white glass (554 mg, 2.021 mmol, 94%). ¹HNMR (270 MHz, CDCl₃) (mixture of imine and methyl ether forms) δ7.72(imine, d, J=4.39, 1H), 7.29 (s, 1H), 3.90 (s, 3H), 3.88-3.51 (m,3H+2H+1H), 2.37-2.04 (m, 4H), 2.22 (s, 3H); ¹³C NMR (67.8 MHz, CDCl₃)δ164.65, 161.41, 156.79, 153.47, 133.87, 126.29, 124.27, 105.71, 60.98,55.80, 55.70, 53.71, 46.70, 29.52, 29.34, 24.13, 9.33.

Example 4

Synthesis of the C8-Amines Synthesis of3-(11-Hydroxy-5-oxo-10-(2,2,2-trichloroethyloxocarbonylamino)-(11aS)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[2,1-a][1,4]diazepin-8-yloxy-2-propenylpropanoate(159) (see FIG. 26)

Nitro Di-acid (153)

14.63 g of (4-carboxy-2-methoxy-5hydroxy-phenoxy)propanoic acid 152 (61mmol) was added portionwise to 70% nitric acid (100 mL ) stirred at 0°C. The reaction was stirred for 1 hour at 0° C. then allowed to returnto rt. The reaction mixture was then poured onto ice and allowed to stirfor 18 h. The solids were then collected by filtration and washed withwater. The aqueous layer was then extracted with ethyl acetate (3×150mL). The organics were then washed with water and brine and dried withsodium sulphate. The solvent was then removed in vacuo to give 153 as ayellow solid, yield=14.01 g (83%) mp 141° C. ¹H NMR (CDCl₃): δ8.51 (bs,2H, COOH), 7.57 (s, 1H, CHCNO₂), 7.15 (s, 1H, CH₃OCCH), 4.35 (t, 2H,J=6.41 Hz, CH₂CH₂O), 3.99 (s, 1H, OCH₃), 2.86 (t, 2H, J=6.41 Hz,CH₂CH₂O). ¹³C-NMR (CDCl₃): δ33.93 (CH₂CH₂O), 56.42 (OCH₃), 65.20(CH₂CH₂O), 108.27 (NO₂CCH), 111.26 (CH₃OCCH), 122.50 (CCOOH),141.14(CNO₂), 149.21 (CH₂CH₂OC.), 152.40 (CH₃OC.), 166.93 (arom. COOH),172.24 (aliph. COOH). IR (Nujol) ν 2860, 2620, 1740, 1720, 1590, 1540,1480, 1390, 1350, 1290, 1230, 1250, 1200, 1060 cm⁻¹. EIMS m/e (relativeintensity): 286 (M^(+.), 20), 241 (10), 213 (100), 169 (20), 152 (5),111 (20), 96 (5), 79 (5), 73 (15), 55 (10). HRMS Calcd. forC₁₁H₁₄NO₈=285.0511 found=285.0538.

2-Propene 3-(4-carboxy-2-methoxy-5-nitrophenoxy)propanoate (154)

A mixture of 3-(4-carboxy-2-methoxy-5-nitrophenoxy)propanoic acid (153)(20 g, 74.3 mmol) and p-toluene sulphonic acid monohydrate (2.3 g, 7.4mmol) in allyl alcohol (240 mL, 3.5 mol) was refluxed for 7 hours thenallowed to cool. The allyl alcohol was then removed in vacuo, and theresidue triturated with dilute HCl acid and collected by filtration.This solid was taken up in EtOAc, and the resulting solution washed withwater and brine and dried over sodium sulphate. Evaporation in vacuoafforded 154 as a white solid (19.27 g, 84%): mp 128-130° C.; ¹H-NMR(CDCl₃): δ2.92 (t, 2H, J=6.35 Hz); 3.94 (s, 3H); 4.38 (t, 2H, J=6.41Hz); 4.65 (d, 2H, J=5.61 Hz); 5.27 (dd, 1H, J₁=1.28 Hz, J₂=19.42 Hz);5.33 (dd, 1H, J₁=1.28 Hz, J₂=17.04 Hz); 5.92 (m, 1H); 7.15 (s, 1H); 7.45(s, 1H); ¹³C NMR (67.8 MHz, CDCl₃): δ34.1, 56.5, 65.0, 65.4, 108.5,111.3, 118.3, 122.9, 131.8, 141.1, 149.1, 152.6, 167.1, 170.0; IR(Nujol); ν 1730, 1630, 1550, 1430, 1390, 1290, 1230, 1190, 1170, 1070,1030, 1010 cm⁻¹; MS (EI) m/z (relative intensity): 325 (M^(+.), 19), 251(3), 213 (2), 196 (3), 211 (3), 113 (19), 91 (4), 71 (9), 55 (6); HRMS:calcd. for C₁₄H₁₅NO₈ 325.0798, found 232.0773.

Prop-2-enyl4-(N-2S-Diethylthiomethylpyrrolidinecarboxy)-2-methoxy-5-nitrophenoxy)propanoate(155)

2-Propene 3-(4-carboxy-2-methoxy-5-nitrophenyloxy)propanoate (154): 5 g,15.34 mmol), oxalyl chloride (2 mL, 23 mmol) and 5 drops of DMF werestirred in dry THF (100 mL) for 18 h. The solvent was then removed invacuo and the residue dissolved in dry THF (50 mL). This was addeddropwise to a vigorously stirred mixture of(2s)-pyrrolidone-2-caroxaldehyde diethyl thioacetate(3.15 g, 15.34 mmol)and triethylamine (1.86 g, 18.41 mmol). The stirring was continued for18 h. The solvent was then removed in vacuo and the product purified byflash chromatography eluting with ethyl acetate to give 155 (7.48 g,95%) as a yellow oil. ¹H NMR (CDCl₃): δ7.74 (s, 1H, OCCHC), 6.83 (s, 1H,MeOCCHC), 5.98-5.86 (m, 1H, CH₂CHCH₂, 5.33 (d, 1H, J=26.56 Hz,OCH₂CHCH₂), 5.28 (d, 1H, J=20.24 Hz, OCH₂CHCH₂), 4.88 (d, 1H, J=3.85 Hz,NCHCH), 4.74-4.65 (m, 2H, OCH₂CHCH₂) 4.42 (t, 2H, J=7.69 Hz, CH₂CH₂OC.),3.94 (s, 3H, OCH₃), 3.29-3.21 (m, 2H, NCH₂), 2.96 (p, 2H, J=3.12 Hz,CH₂CH₂O), 2.87-2.67 (m, 4H, SCH₂CH₃), 2.32-1.78 (m, 4H, NCH₂CH₂CH₂)1.38-1.31 (m, 6H, SCH₂CH₃). ¹³C-NMR (CDCl₃): δ15.00, 15.13 (SCH₂CH₃),24.63 (NCH₂CH₂CH₂), 26.28, 26.59, 27.22 (NCH₂CH₂CH₂), 34.13 (CH₂CH₂O),50.19 (NCH₂), 52.80 (NCHCH), 56.60 (OCH₃), 61.08 (NCH), 65.13 (CH₂CH₂O),65.64 (OCH₂CHCH₂), 108.70 (arom. CH), 109.47 (arom. CH), 118.55(OCH₂CHCH₂), 128.58 (CCON), 131.73 (OCH₂CHCH₂), 137.17 (CNO₂), 147.98(CH₂CH₂OC.), 154.57 (COCH₃), 166.61 (CON), 170.14 (COO). IR (Nujol)ν=3550-2720, 3000, 2630, 2200, 1740, 1640, 1580, 1530, 1340, 1280, 1220,1180, 1050 cm⁻¹. MS (EI): m/e (relative intensity): 527 (M^(+.), 1), 377(10), 310 (12), 309 (72), 308 (94), 268 (20), 142 (4). HRMS calcd. forC₂₄H₃₅O₇N₂S₂=527.1875, found=527.1885.

5-Amino-3-(4-(2-diethylthiomethyl-(2S)-perhydro-1-pyrroloylcarbonyl)-2-methoxyphenyloxy)₂-propenylpropanoate(156)

8 (7.21 g, 14.05 mmol) and Tin(II) chloride (15.85 g, 76 mmol) wasrefluxed for 40 minutes in ethyl acetate (100 mL) then allowed to cool.The solvent was then removed in vacuo and the residue was trituratedwith saturated bicarbonate solution at 0° C. EtOAc (50 mL) was added andthe reaction stirred overnight. The reaction mixture was then filteredthrough Celite and the filter cake washed with ethyl acetate. Thecombined organics were then washed with water and brine, dried withsodium sulphate and the solvent removed in vacuo. The product waspurified using flash chromatography eluting with 5% MeOH indichloromethane to give a yellow oil, yield=5.87 g (86%). ¹H NMR(CDCl₃): δ6.82 (s, 1H, arom. CH), 6.28 (s, 1H, arom.CH), 5.99-5.85 (m,1H, OCH₂CHCH₂), 5.31 (dd, 1H, J=1.28 Hz, 27.66 Hz, OCH₂CHCH₂), 5.26 (dd,1H, J=1.28 Hz, 20.70 Hz, OCH₂CHCH₂), 4.71-4.62 (m, 5H, including doubletat 4.62, 2H, J=5.49 Hz, NH₂+NCHCH, OCH₂CHCH₂), 4.27 (t, 2H, J=6.59 Hz,CH₂CH₂O), 3.92, (m, 1H, NCH), 3.74 (s, 3H, OCH₃), 3.66-3.57 (m, 2H,NCH₂) 2.89 (t, 2H, J=6.6 Hz, CH₂CH₂O), 2.83-2.64 (m, 4H, SCH₂CH₃),2.28-1.80 (m, 4H, NCH₂CH₂CH₂), 1.25 (m, 6H, SCH₂CH₃); ¹³C NMR (CDCl₃)δ14.20 (SCH₂CH₃), 26.55, 27.23 (NCH₂CH₂CH₂), 34.27 (CH₂CH₂O), 53.20(NCHCH), 56.08 (OCH₃), 60.10 (NCH), 60.39 (NCH₂), 64.20 (CH₂CH₂O), 64.41(OCH₂CHCH₂), 102.26 (arom. CH), 113.71 (arom. CH), 118.40 (OCH₂CHCH₂),131.93 (OCH₂CHCH₂), 141.03 (CNH₂), 141.74 (CH₂CH₂OC.), 154.56 (COCH₃),169.69 (CON), 170.53 (COO). IR (neat liquid film) 3500-3000, 3460, 3400,2970, 1740, 1650, 1535, 1470, 1345, 1290, 1225, 1190 cm⁻¹; MS (EI): m/e(relative intensity): 482 (M^(+.), 4), 347 (2), 278 (31), 137 (1), 70(3); HRMS calcd. for C₂₃H₃₄O₅N₂S₂=482.1909, found=482.1925.

3-(4-(2-Diethylthiomethyl-(2S)-perhydro-1-pyrrolylcarbonyl)-2-methoxy-5-(2,2,2-trichloroethyloxycarbonylamino)phenyloxy)₂-propenylpropanoate(157)

To a solution of 156 (5.67 g, 11.74 mmol) in dichloromethane (200 mL)was added pyridine (2.02 mL, 23.48 mmol). To this was added dropwise at0° C. a solution of trichloroethyl chloroformate (1.616 mL, 11.74 mmol).The solution was stirred for a further 1 hour at 0° C. The organics werewashed with 1 N HCl (3×100 mL), water (3×100 mL) brine (100 mL), driedover magnesium sulphate and the solvent removed in vacuo to give a brownoil (6.8 g, 88%) ¹H NMR (CDCl₃): δ9.14 (bs, 1H, NH), 7.88 (bs, 1H,CHCNH), 6.93 (s, 1H, MeOCCHC), 5.99-5.86 (m, 1H, OCH₂CHCH₂), 5.31 (dt,1H, J=1.47 Hz, 27.84 Hz OCH₂CHCH₂), 5.25 (dt, 1H, J=1.29 Hz, 21.61 Hz,CH₂CHCH₂),4.89-4.77 (m, 4H, including doublet 1H, J=1.28 Hz, CHCHSEt,NH, CH₂-TrOC), 4.62 (d, 2H, J=1.28 Hz, OCH₂CHCH₂), 3.81 (s, 3H, OCH₃),3.60 (m, 2H, NCH₂), 2.91 (d, 2H, J=6.42 Hz, CH₂CH₂O), 2.84-2.61 (m, 4H,SCH₂CH₃), 1.37-1.23 (m, 6H, SCH₂CH₃); ¹³C NMR (CDCl₃): δ170.33 (esterCO), 168.50 (CON), 151.94 (OCO), 150.29 (COCH₃), 144.52 (COCH₂CH₂),131.93 (OCH₂CHCH₂), 131.35 (CNH), 118.29 (OCH₂CHCH₂), 112.21 (arom. CH),105.51 (arom. CH), 95.27 (CCl₃), 76.24 (CH₂TrOC), 74.39 (CH₂TrOC), 65.42(CH₂CH₂O), 61.14 (NCH), 56.30 (OCH₃), 53.00 (NCHCHSEt), 34.27 (CH₂CH₂O),27.30, 26.71, 26.43, 25.24 (NCH₂CH₂CH₂), 15.27, 14.87, 14.18 (SCH₂CH₃).MS (EI): m/e (relative intensity): 658, 656 (M^(+.), 1), 508 (1), 373(6), 305 (5), 304 (27), 192 (5), 70 (12).

3-(11-Hydroxy-5-oxo-10-(2,2,2-trichloroethyloxocarbonylamino)-(11aS)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[2,1-a][1,4]diazepin-8-yloxy-2-propenylpropanoate(158)

A solution of 157 (6.8 g, 10.34 mmol) in acetonitrile/water (4:1, 200mL) was treated with calcium carbonate (2.585 g, 25.85 mmol) andmercuric(II) chloride (7.00 g, 25.85 mmol) and the solution was stirredfor 18 h. The reaction was then filtered through Celite and the filterpad washed with ethyl acetate. The organics were collected and washedwith water (3×50 mL), brine (100 mL) and dried over magnesium sulphate.The solvent was removed in vacuo and the resulting product was purifiedby flash chromatography eluting with ethyl acetate to give the productas a yellow oil (3.67 g, 64%) ¹H NMR (CDCl₃): δ7.25 (arom. CH), 6.86 (s,1H, arom. CH), 6.00-5.85 (m, 1H, CH₂CHCH₂), 5.67 (d, 1H, J=9.71 Hz,TrOC—CH₂) 5.37-5.20 (m, 3H, TrOC—CH₂+OCH₂CHCH₂), 4.65 (d, 2H, J=5.67 Hz,CH₂CHCH₂O), 4.36-4.22 (m, 3H, CH₂CH₂O+NCHOH), 3.90 (s, 3H, OCH₃),3.72-3.47 (m, 3H, NCH+NCH₂), 2.91 (t, J=6.41 Hz, CH₂CH₂O) 2.29-2.00 (m,4H, NCH₂CH₂CH₂) ¹³C NMR (CDCl₃): δ170.33 (ester carbonyl CO), 166.17(CON), 154.4 (OCO), 149.88 (COCH₃), 148.93 (COCH₂CH₂), 131.86(CH₂CHCH₂), 127.48 (arom. CN), 126.24 (CCON), 118.42 (OCH₂CHCH₂), 114.48(arom. CH), 110.82 (arom. CH), 95.09 (CCl₃), 86.42 (NCHOH), 74.96(TrOC—CH₂), 65.47 (OCH₂CHCH₂), 64.43 (CH₂CH₂O), 60.13 (NCH), 56.14(OCH₃), 46.44 (NCH₂), 34.26 (CH₂CH₂O), 28.64 (NCH₂CH₂CH₂),MS (EI) m/z(relative intensity): =552 (M⁺10), 550 (10), 374 (2), 368 (5), 304 (15),192 (8), 70 (24), 55(24). HRMS calcd. for C₂₂H₂₅N₂O₈Cl₃=552.0651, found3 peaks due to chlorine 552.0646, 550.676, 554.0617.

3-(11-Hydroxy-5-oxo-7-methoxy-10-(2,2,2-trichloroethyloxocarbonylamino)-(11aS)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[2,1-a][1,4]diazepin-8-yloxypropanoicacid (159)

A solution of 158 (3.5 g, 6.35 mmol) was dissolved in ethanol (100 mL).To this was added Tetrakis(triphenylphospine)palladium(0) (350 mg, 0.303mmol) and the solution refluxed for 30 minutes until the reaction wascomplete by TLC monitoring. The reaction was then allowed to cool andthe filtered through Celite. The EtOH was then removed in vacuo to givethe crude material as a yellow solid which was used directly in the nextsteps. ¹H-NMR (CDCl₃): δ7.22 (s, 1H, OCCHCN), 7.01 (s, 1H, MeOCCHC),6.27 (bs, COOH), 5.67 (d, 1H, J=9.5 Hz, TrOC—CH₂), 5.06 (d, 1H, J=12.09Hz, TrOC—CH₂), 4.29-4.11 (m, 2H, CHOH), 3.85 (s, 3H, OCH₃), 3.71 (t, 2H,J=6.97 Hz, CH₂CH₂O), 3.51 (m, 1H, NCH), 2.80 (m, 2H, NCH₂), 2.12-1.99(m, 4H, NCH₂CH₂CH₂), 1.21 (t, 2H, J=6.96 Hz, CH₂CH₂O) ¹³C NMR (CDCl₃):δ=174.27 (acid CH), 167.34 (CON), 154.20 (OCO), 149.78 (COCH₃), 148.74(COCH2CH2), 133.79 (arom. CH), 132.16 (arom. CH), 128.66 (arom. CN),125.87 (CCON), 95.06 (CCl₃), 86.53 (NCHCHOH), 74.95 (CH₂-TrOC), 60.67(NCH), 58.24 (CH,CH₂O), 56.04 (OCH₃), 46.44 (NCH₂), 35.24 (NCH₂CH₂CH₂),28.59 (NCH₂CH₂CH₂), 23.08 (CH₂CH₂O)

Example 4(a)

3-(7-methoxy-5-oxy(11aS)-2,3,5,11a-tetrahydro-1H-benzo[e]pyrro[1,2-a[]1,4]diazepin-8-yloxy)-1-perhydro-1-pyrrolyl-1-propanone(161)(see FIG. 27)

3-(11-Hydroxy-7-methoxy-5-oxo-10-(2,2,2-trichloroethyloxocarbonylamino)-(11aS)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[2,1-a][1,4]diazepin-8-yloxy-1-perhydro-1-pyrrolyl-1-propanone(160)

To a solution of 159 (100 mg, 0.196 mmol) in dichloromethane was added1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (44 mg, 0.23mmol) and 4-(dimethylamino)pyridine (5 mg, 0.04 mmol) and the solutionstirred for 1 h. To the reaction was added pyrrolidine (16.36 mg, 0.23mmol) and the reaction stirred for a further 2 h. The solvent was thenremoved in vacuo and the compound purified by flash chromatographyeluting with 5% methanol in dichloromethane to give the product as ayellow oil, yield=56 mg, 51%. ¹H NMR (CDCl₃) δ7.25 (OCCH), 6.90 (s, 1H,MeOCCHC), 5.66 (d, 1H, J=5.49 Hz, TrOC—CH₂), 5.16 (d, 1H, J=12.09 Hz,TrOC—CH₂), 4.84-4.74 (m, 2H, CHOH, C11aH), 4.35-4.23 (m, 2H, CH₂CH₂O),3.90 (s, 3H, OCH₃), ), 3.73-3.67 (m, 1H, NCH), 3.53-3.44 (m, 6H C-ringNCH₂, pyrrolidine-N(CH₂)₂), 2.92-2.76 (m, 2H CH₂CH₂O), 2.11-1.85 (8H,C-ring NCH₂CH₂CH₂+pyrrolidine-NCH₂CH₂CH₂); ¹³C-NMR (CDCl₃): δ168.62(amide CO), 167.05 (CON), 154.31 (OCO), 149.94 (COCH₃), 148.56(COCH₂CH₂), 127.76 (arom. CN), 125.95 (CCON), 114.14 (arom. CH), 110.49(arom. CH), 95.04 (CCl₃), 86.48 (NCHCHOH), 74.98 (CH₂—TROC), 65.15(CH₂CH₂O), 60.20 (NCH), 56.13 (OCH₃), 46.85, 46.44, 45.76, 34.47, 28.60,26.02, 24.42 (various N—(X)CH₂), 23.04 (CH₂CH₂O); FABMS m/z (relativeintensity) 564 (M⁺1), 550(3), 549 (2), 548 (8), 547 (2), 546 (8), 279(2), 192 (4), 126 (18), 98 (6).

3-(7-methoxy-5-oxy(11aS)-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)-1-perhydro-1-pyrrolyl-1-propanone(161)

Method A: To a solution of 160 (100 mg, 0.164 mmol) in dichloromethane(5 mL) was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (38 mg, 0.2 mmol) and pyrrolidine (14 mg, 0.2 mmol) andthe reaction stirred for 18 h. The mixture was then dilute withdichloromethane (100 mL) and washed with water (3×50 mL), saturatedsodium bicarbonate solution (3×50 mL) and brine (50 mL). The solvent wasremoved in vacuo and the product purified by flash chromatographyeluting with 5% methanol in dichloromethane to give the product 161 as awhite solid (yield 26.3 mg, 40%)

Method B: To a solution of 160 (100 mg, 0.164 mmol) in dichloromethane(5 mL) was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (38 mg, 0.2 mmol) and the reaction stirred for 3 hours.The reaction was then treated with tetrabutylammonium fluoride (200 μLof a 1.0 M solution in THF, 0.2 mmol) and stirred for 30 minutes. Thereaction was then treated with pyrrolidine (14 mg, 0.2 mmol) and stirredfor 18 h. The mixture was then dilute with dichloromethane (100 mL) andwashed with water (3×50 mL), saturated sodium bicarbonate solution (3×50mL) and brine (50 mL). The solvent was removed in vacuo and the productpurified by flash chromatography eluting with 5% methanol indichloromethane to give the product 161 as a white solid (yield 54.2 mg,82%)

Method C: To a solution of 160 (56 mg, 0.1 mmol) in THF (3 mL) was added1 N ammonium acetate solution (2 mL) and the reaction mixture stirred.To the solution was added 10% Cd/Pb couple (0.5 mmol, 62.4 mg) and thereaction was stirred for 90 minutes. The reaction was filtered anddiluted with ethyl acetate (20 mL). The solution was dried withmagnesium sulphate and the solvent removed in vacuo. the product as thenpurified by flash chromatography eluting with 5% methanol indichloromethane to give the compound as a white solid (yield =21 mg,56%). ¹H NMR (CDCl₃): δ7.66 (m, 1H, J=4.39 Hz, N═CH), 7.50 (s, 1H, arom.CH), 6.88 (s, 1H arom. CH), 4.42 (t, 2H, J=6.96 Hz, OOCCH₂CH₂), 3.92 (s,3H, OCH₃), 3.90-3.44 (m, 5H, pyrrolidine CH₂+NCH), 2.87 (t, 2H, 5.96 Hz,OOCCH₂CH₂), 2.28-2.33 (m, 2H, NCH₂CH₂), 2.10-1.87 (m, 8H,C-ring+pyrrolidine CH₂). 168.58 (amide CO), 164.65 (CON), 162.43 (imineCH), 150.52 (COCH₃), 147.61 (COCH₂CH₂), 140.76 (arom. CN), 120.33(CCON), 111.54 (arom. CH), 110.61 (arom. CH), 65.20 (COCH₂CH₂), 56.21(COCH₃), 53.7 (NCH), 46.77, 46.67, 45.69, 34.40, 29.62, 26.06, 24.54,(CH₂), 24.19 (COCH₂CH₂) MS (EI): m/e (relative intensity): 371 (M^(+.),10), 246 (10), 245 (5), 231 (3), 126 (18), 98 (2), 70 (5), 55 (3); HRMScalcd. for C₂₀H₁₅O₄N₃=371.1845, found 371.1788.

Example 4(b)

3-(7-methoxy-5-oxy(11aS)-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4diazepin-8-yloxy)-1-piperidino-1-propanone(163) (see FIG. 27)

3-(11-Hydroxy-7-methoxy-5-oxo-10-(2,2,2-trichloroethyloxocarbonylamino)-(11aS)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[2,1-a][1,4]diazepin-8-yloxy-1-perhydro-1-piperidino-1-propanone(162)

To a solution of 159 (100 mg, 0.196 mmol) in dichloromethane was added1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (44 mg, 0.23mmol) and 4-(dimethylamino)pyridine (5 mg, 0.04 mmol) and the solutionstirred for 1 h. To the reaction was added piperidine (25 μL, 0.23 mmol)and the reaction stirred for a further 2 h. The solvent was then removedin vacuo and the compound purified by flash chromatography eluting with5% methanol in dichloromethane to give the product as a yellow oil,yield=94 mg, 84%). ¹H-NMR (CDCl₃): δ7.25 (s, 1H, OCCHCN), 6.90 (s, 1H,MeOCCHC), 5.65 (d, 1H, J=9.71 Hz, TrOC—CH₂), 5.17 (d, 1H, J=11.94 Hz,TrOC—CH₂), 4.37-4.24 (m, 4H, CHOH+CH₂CH₂O), 3.91 (s, 3H, OCH₃),3.73-3.67 (m, 1H, NCH), 3.54-3.45 (m, 6H, NCH₂, piperidine-N(CH₂)₂),2.99-2.83 (m, 2H, CH₂CH₂O), 2.13-2.00 (m, 4H, NCH₂CH₂CH₂) 1.67-1.56 (m,6H, piperidine-CH₂); ¹³C NMR (CDCl₃): δ168.22 (amide CO), 167.11 (CON),154.38 (OCO), 149.96 (COCH₃), 148.57 (COCH₂CH₂), 127.74 (arom. CN),125.94 (CCON), 114.19 (arom. CH), 110.44 (arom. CH), 95.02 (CCl₃), 86.38(NCHCHOH), 74.96 (CH₂—TROC), 65.38 (CH₂CH₂O), 60.33 (NCH), 56.08 (OCH₃),46.77 , 46.44, 42.75, 32.73, 28.60, 26.33, 25.48, 24.44 (variousN—(X)CH₂), 23.05 (CH₂CH₂O); MS (EI) m/z (relative intensity): =579 (1),577 (1), 331 (1), 278 (1), 246 (1), 192 (4), 140 (32), 113 (2), 112 (2),97 (1), 84 (3), 77 (3), 70 (7), 69 (4), 55 (4), HRMS calcd. forC₂₄H₃₀N₃O₇Cl₃=579.1120 found 579.1066

3-(7-methoxy-5-oxy(11aS)-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)-1-piperidino-1-propanone(163)

To a solution of 162 (94 mg, 0.162 mmol) in THF (3 mL) was added 1 Mammonium acetate solution (2 mL) and the reaction mixture stirred. Tothe solution was added 10% Cd/Pb couple (0.81 mmol, 100 mg) and thereaction was stirred for 90 minutes. The reaction was filtered anddiluted with ethyl acetate (20 mL). The solution was dried withmagnesium sulphate and the solvent removed in vacuo. the product as thenpurified by flash chromatography eluting with 5% methanol indichloromethane to give the compound as a white solid (yield 25 mg,39%). ¹H NMR (CDCl₃): δ7.67 (d, 1H, J=4.4 Hz, N═CH), 7.51 (s, 1H,OCCHCN), 6.89 (s, 1H, MeOCCHC), 4.42 (t, 2H, J=7.14 Hz, CH₂CH₂O), 3.93(s, 3H, OCH₃), 3.90-3.44 (m, 5H, NCH, NCH₂, piperidine-N(CH₂)₂), 2.73(t, 2H, J=7.32 Hz CH₂CH₂O), 2.33-2.29 (m, 2H, C-ring CH₂), 2.11-2.02 (m,2H, C-ring CH₂), 1.62-1.59 (m, 6H, piperidine CH₂), ¹³C NMR (CDCl₃):δ168.19 (amide CO), 164.66 (imine CH), 162.43 (CON), 150.52 (COCH₃),147.61 (COCH₂CH₂), 140.70 (arom. CN), 120.31 (CCON), 111.51 (arom. CH),110.58 (arom. CH), 65.44 (CH₂CH₂O), 56.11 (OCH₃), 53.73 (NCH), 46.70,46.39, 42.69, 32.72, 29.62, 26.38, 25.52, 24.40 (various N—(X)CH₂),24.19 (CH₂CH₂O); MS (EI): m/e (relative intensity): 385 (M^(+.), 6), 246(8), 245 (3), 231 (3), 140 (15), 138 (5), 97 (5). 84 (3); HRMS calcd.for C₂₁H₂₇O₄N₃=385.2002, found 385.2058.

Example 4(c)

1,(2,3-dihydro-1H-indolyl)-3-(7-methoxy-5-oxy(11aS)-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)-1-propanone(165) (see FIG. 27)

1-(2,3-Dihydro-1H-1-indolyl)-3-(11-hydroxy-7-methoxy-5-oxo-10-(2,2,2-trichloroethyloxocarbonylamino)-(11aS)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[2,1-a][1,4]diazepin-8-yloxy-1-propanone(164)

To a solution of 159 (100 mg, 0.196 mmol) in DMF was added1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (44 mg, 0.23mmol) and 4-(dimethylamino)pyridine (5 mg, 0.04 mmol) and the solutionstirred for 1 h. To the reaction was added indoline (27.4 mg, 0.23 mmol)and the reaction stirred for a further 8 h. The solvent was then removedin vacuo and the compound purified by flash chromatography eluting with5% methanol in dichloromethane to give the product as a yellow oil(yield=71 mg, 61%). ¹H-NMR (CDCl₃): δ1.99-2.12 (m, 4H, NCH₂CH₂CH₂), 3.20(t, J=8.42 Hz, CH₂CH₂O), 3.71-5.00, (m, 4H, NCH₂, NCH, CHOH), 3.89 (s,3H, OCH₃), 4.18-4.09 (m, 2H, indole-CH₂), 4.27 (d, 2H, J=11.90 Hz,indole-CH₂), 4.43 (t, J=6.23 Hz, CH₂CH₂O), 5.16 (d, 1H, J=11.91 Hz,TrOC—CH₂), 5.30 (s, 1H, OH), 5.66 (d, 1H, J=9.89 Hz, TrOC—CH₂),7.20-6.93 (m, 5H, indole-CH, arom CH), 8.18 (d, 1H, J=8.25 Hz,indole-CH); ¹³C-NMR (CDCl₃): δ168.24 (CON), 166.97 (CON), 154.36 (OCO),149.91, COCH₃), 148.65 (COCH₂CH₂), 132.14, 131.99 (indolyl ringjunction), 128.61, 128.43 (indole-CH), 127.52, (arom. CN), 124.61(CCON), 114.20 (arom. CH), 110.58 (arom. CH) 95.02 (CCl₃), 86.43(NCHCHOH), 75.01 ((TrOC—CH), 64.89 (CH₂CH₂O), 60.13 (NCH), 56.11 (OCH₃),48.11 (indole-CH₂), 46.43 (NCH₂), 35.64, 28.64, 27.97, (CH₂), 23.03(CH₂CH₂O); MS (EI) m/z (relative intensity): =595 (M⁺1), 415 (1), 365(1), 246 (2), 192 (13), 174 (11), 173 (7), 119 (17), 118 (10), 70 (13).

Iso-indoline (2,3,-dihydro-1H-isoindole)

¹H NMR (CDCl₃): δ7.22 (m, 4H, arom CH), 4.26 (s, 4H, CH₂), 4.08 (bs, 1H,NH), ¹³C NMR (CDCl₃): δ140.37, 140.36 (ring junctions), 127.15, 126.90,122.60, 122.51, 122.33 (arom. CH), 52.31 (CH₂).

1,(2,3-dihydro-1H-indolyl)-3-(7-methoxy-5-oxy(11aS)-2,3,5,11a-tetrahydro-1H-benzo]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)-1-propanone(165)

To a solution of 164 (71 mg, 0.116 mmol) in THF (3 mL) was added 1 Mammonium acetate solution (2 mL) and the reaction mixture stirred. Tothe solution was added 10% Cd/Pb couple (0.58 mmol, 72 mg) and thereaction was stirred for 90 minutes. The reaction was filtered anddiluted with ethyl acetate (20 mL). The solution was dried withmagnesium sulphate and the solvent removed in vacuo. The product as thenpurified by flash chromatography eluting with 5% methanol indichloromethane to give the compound as a white solid (yield=26 mg,54%). ¹H NMR (CDCl₃): δ7.66 (d, 1H, J=4.58 Hz, CH═N), 7.50 (s, arom.CH), 7.19 (m, 4H indolyl arom. CH), 6.91 (s, 1H, arom. CH), 4.48 (m, 2H,CH₂CH₂O), 4.18-4.19 (m, 2H, indolyl CH₂), 3.91 (s, 3H, OCH₃), 3.88-3.44(m, 3H, NCH, +indolyl CH₂), 3.02 (t, 2H, J=6.6 Hz, CH₂CH₂O), 2.30-2.28(m, 2H, NCH₂), 2.17-2.05 (m, 4H, NCH₂CH₂CH₂); ¹³C NMR (CDCl₃): δ168.31(amide CO), 164.61 (CON), 162.47, (imine CH), 147.59 (COCH₂CH₂), 140.70(arom. CN), 127.53, 124.59, 123.87, (indolyl arom. CH), 120.44 (CCON),117.03 (indolyl arom. CH), 11.56 (arom. CH), 110.61 (arom. CH), 64.80(COCH₂CH₂), 56.14 (COCH₃), 53.70 (NCH), 48.11, 46.69, 35.50, 29.60,28.67, 28.00 (CH₂), 24.19 (COCH₂CH₂).

Example 4(d)

1,(2,3-dihydro-1H-2-isoindolyl)-3-(7-methoxy-5-oxy(11aS)-2,3,5,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)-1-propanone(167) (see FIG. 27)

1-(2,3-dihydro-1H-2-isoindolyl)-3-(11-hydroxy-7-methoxy-5-oxo-10-(2,2,2-trichloroethyloxocarbonylamino)-(11aS)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[2,1-a][1,4]diazepin-8-yloxy-1-propanone(166)

To a solution of 159 (100 mg, 0.196 mmol) in DMF was added1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (44 mg, 0.23mmol) and 4-(dimethylamino)pyridine (5 mg, 0.04 mmol) and the solutionstirred for 1 h. To the reaction was added indoline (27.4 mg, 0.23 mmol)and the reaction stirred for a further 8 h. The solvent was then removedin vacuo and the compound purified by flash chromatography eluting with5% methanol in dichloromethane to give the product as a yellow oil(yield =75 mg, 64%). ¹H-NMR (CDCl₃): δ7.29-7.20 (m, 5H, isoindolearom.+arom.CH), 6.91 (s, 1H, arom CH), 5.66 (d, 1H, J=9.7 Hz, TrOC—CH₂)5.30 (s, 1H, OH), 5.19 (d, 1H, J=9.7 Hz, TrOC—CH₂), 4.94 (m, 2H,isoindolyl CH₂), 4.79 (s, 2H, isoindolyl CH₂), 4.38 (t, 2H, J=6.42 Hz,CH₂CH₂O), 4.25, (d, 1H, J=11.91 Hz, C11-H), 3.81-3.40 (2H, NCH₂),3.03-2.85 (m, 2H, CH₂CH₂O), 2.11-1.98 (m, 4H, NCH₂CH₂CH₂); ¹³C-NMR(CDCl₃): δ169.17 (CON), 167.02 (CON), 154.27 (OCO), 149.91 (COCH₃),148.64 (COCH₂CH₂), 136.19, 136.11 (isoindolyl ring junction), 128.61,127.88 (isoindolyl CH), 127.78 (arom. CN), 127.58, (CCON), 114.28 (arom.CH), 110.54 (arom. CH), 95.09 (CCl₃), 86.51 (NCHCHOH), 74.98 (TrOC—CH₂),65.21 (CH₂CH₂O), 60.23 (NCH), 56.05 (OCH₃), 52.14, 52.81 (isoindolylCH₂), 46.43, (NCH₂), 34.31, 29.68, 28.60 (NxCH₂), 23.03 (CH₂CH₂O); FABMSm/z (relative intensity): =612 (1), 596 (1), 594 (1), 279 (1), 192 (1),174 (8), 146 (5), 118 (13), 91 (2), 55 (3). FABHRMS found compound minusOH i.e. C₂₇H₂₇N₃O₆Cl₃=595.1044

1,(2,3-dihydro-1H-2-isoindolyl)-3-(7-methoxy-5-oxy(11aS)-2,3,5,11a-tetrahydro-1H-benzo]pyrrolo[1,2-a][1,4]diazepin-8-yloxy)-1-propanone(167)

To a solution of 166 (75 mg, 0.122 mmol) in THF (3 mL) was added 1 Mammonium acetate solution (2 mL) and the reaction mixture stirred. Tothe solution was added 10% Cd/Pb couple (0.61 mmol, 76 mg) and thereaction was stirred for 90 minutes. The reaction was filtered anddiluted with ethyl acetate (20 mL). The solution was dried withmagnesium sulphate and the solvent removed in vacuo. The product wasthen purified by flash chromatography eluting with 5% methanol indichloromethane to give the compound as a white solid (yield=42.6 mg,83%). ¹H NMR (CDCl₃): δ7.66 (d, 2H, J=4.39 Hz, N═CH), 7.48 (s, 1H, arom.CH), 7.30 (s, 4H, indolyl arom. CH), 6.89 (s, 1H, arom. CH), 4.48 (t,3H, J=6.59 Hz, COCH₂CH₂), 3.84 (s, 3H, OCH₃), 3.81-3.69 (m, 2H, indolylCH₂), 3.61-3.51 (m, 1H, NCH), 2.97 (p, 5H, J=6.9 Hz, CH₂CH₂O), 2.32-2.28(m, 2H, NCH₂), 2.30-2.01 (m, 4H, NCH₂CH₂CH₂); ¹³C NMR (CDCl₃): δ169.29(amide CO), 164.66 (imine CH), 162.52 (CON), 150.45 (COCH₃), 147.63(COCH₂CH₂), 140.57, (arom. CN), 127.86, 127.56, 123.04, 122.62 (indolylarom. CH), 120.38 (CCON), 111.52 (arom. CH), 110.53 (arom. CH), 65.16(COCH₂CH₂), 56.06 (COCH₃), 53.73 (NCH), 52.16, 50.64, 46.70, 34.22,29.57 (CH₂), 24.18 (COCH₂CH₂); MS (EI): m/e (relative intensity): 419(M^(+.), 21), 416 (2), 415 (2), 246 (10), 245 (3), 231 (3), 174 (4);HRMS calcd. for C₂₄H₂₅O₄N₃=419.1845, found 419.1821.

Example 4(e)

Synthesis of (11aS)8-(N-9-fluorenylmethoxycarbonyl)aminopropyloxy-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine-5-one(205) (See FIG. 25)

Synthesis of N-(tert-butoxycarbonyl)-3-hydroxypropylamine (196)

A solution of (Boc)₂O (25.0 g, 114.5 mmol) in anhydrous DCM (50 mL) wasadded dropwise to a stirred solution of 3-amino-1-propanol (195) (7.8 g,104.5 mmol) in anhydrous DCM (100 mL), under a nitrogen atmosphere. Thereaction mixture was allowed to stir for 12 hours, after which time TLC(50% pet-ether/EtOAc) revealed complete loss of starting material. Thesolution was diluted with Et₂O (150 mL) and washed with phosphate buffer0.5 M, pH5.4 (2×70 mL), sat. aqueous NaHCO₃ (70 mL), brine (2×70 mL) anddried over MgSO₄. Excess solvent was removed by evaporation underreduced pressure to give a viscous colourless oil (196) (18.3 g, 100%).¹H NMR (270 MHz, CDCl₃): δ1.44 (s, 9H, CH₃), 1.67 (m, 2H, H2′), 3.26 (q,2H, J=6.23 Hz, H3′), 3.65 (dd, 2H, J=5.86, 5.68 Hz, H1′), 3.78 (dt, 1H,J=6.04, 5.87 Hz, OH), 5.18 (br, 1H, NH); ¹³C NMR (67.8 MHz, CDCl₃):δ28.4 (CH₃), 32.6 (C2′), 37.1 (C3′), 59.3 (C1′), 79.4 (C_(quater)),157.1 (C═O); MS (E/I) m/z (relative intensity): 176 (M^(+.), 30), 120(100), 119 (31), 102 (49), 83 (33), 76 (67), 74 (36); HRMS (E/I) exactmass calcd for C₈H₁₇O₃N: m/e 175.1200, obsd m/e 175.1208; IR (Nujol^(ò))n: (cm⁻¹) 3355, 2976, 2936, 2878, 1810, 1694, 1531, 1455, 1392, 1366,1278, 1253, 1173, 1072, 996, 914, 870, 781, 752, 638.

Synthesis of Methyl4-[N-(tert-butoxycarbonyl)]aminopropyloxy-3-methoxybenzoate (198)

A solution of DEAD (18.3 g, 105.3 mmol) in freshly distilled THF (50 mL)was added dropwise to a mechanically stirred solution oftriphenylphosphine (27.6 g, 105.3 mmol), methyl vanillate 197 (19.2 g,105.3 mmol), and Boc-amino-1-propanol (196) (18.4 g, 105.3 mmol) infreshly distilled THF (250 mL), at 0° C. under a nitrogen atmosphere.After the DEAD was added the reaction mixture was allowed to stir atroom temperature overnight and the progress of the reaction wasmonitored by TLC (50% EtOAc/pet-ether). The solvent was removed byevaporation under reduced pressure and the residue was triturated withEt₂O (300 mL) to precipitate some of TPO and diethylhydrazinedicarboxylate, which were removed by filtration. The filtratewas washed with 1 N aqueous NaOH (150 mL), H₂O (2×150 mL), brine (2×150mL) and dried over MgSO₄. Excess solvent was removed by evaporationunder reduced pressure and the crude product (198) was purified bycolumn chromatography (80% pet-ether/EtOAc) to afford a beige solid (30g, 85%). mp=79-82° C.; ¹H-NMR (CDCl₃, 270 MHz): δ1.46 (s, 9H, CH₃),2.0-2.08 (m, 2H, H2′), 3.38 (dd, 2H, J=5.68, 6.04 Hz H3′), 3.90 (s, 3H,OCH_(3ester)), 3.93 (s, 3H, OCH_(3ether)), 4.14 (t, 2H, J=5.95 Hz, H3′),5.58 (br, 1H, NH), 6.86 (d, 1H, J=8.42 Hz, _H5), 7.55 (d, 1H, J=1.83 Hz,H2), 7.65 (dd, 1H, J=2.02, 8.42 Hz, H6); ¹³C-NMR (CDCl₃, 68.7 MHz):δ28.5 (C_(prima)), 29.2 (C2′), 38.9 (C3′)) 52.0 (OCH_(3ester)), 55-8(OCH_(3ether)), 68.1 (C1′), 78.9 (C_(quater)), 111.3 (C5), 112.0 (C2),122.84 (C_(arom)), 123.5 (C6), 148.8 (C_(arom)), 152.1 (C_(arom)), 156.1(NC═O), 166.8 (C═O); MS (E/I) m/z (relative intensity): 339 (M^(+.),11), 266 (13), 182 (42), 151 (27), 102 (100); HRMS (E/I) exact masscalcd for C₁₇H₂₅NO₆,: m/e 339.1682, obsd m/e 339.1733; IR (Nujol^(ò)) n:(cm⁻¹) 3362, 2923, 2854, 1712, 1684, 1599, 1520, 1464, 1377, 1272, 1217,1132, 1045, 1022, 872, 780, 762, 722.

Synthesis of Methyl 4-Aminopropyloxy-5-methoxy-2-nitrobenzoate (199)

The ester 198 (4.0 g, 11.8 mmol) was added in small portions to astirred solution of 70% HNO₃ (2 mL acid/g of substrate) at roomtemperature and the reaction mixture was allowed to stir overnight.After 16 hours TLC (CHCl₃) revealed the complete loss of startingmaterial. The reaction mixture was cooled in an ice bath, and 15 g oficed water was added, precipitating the product. The precipitate wascollected by vacuum filtration and washed with small amount of icedwater. The filtrate was cooled and a second crop of precipitate wascollected by vacuum filtration and washed with iced water. The combinedprecipitate was dried in vacuo to provide compound 199 as a yellowsolid, which was not purified further, but used directly in thesubsequent reaction (2.3 g, 70%). mp=101-103° C.; ¹H-NMR (CDCl₃/DMSO-d₆,270 MHz): δ2.31 (m, 2H, H2′), 3.20 (br, 2H, H3′), 3.95 (s, 3H,OCH_(3ether)), 3.98 (s, 3H, OCH_(3ester)), 4.24 (t, 2H, J=5.95 Hz, H1′),7.11 (s, 1H, H6), 7.49 (s, 1H, H3), 8.21 (s, 3H, NH); ¹³C-NMR (CDCl₃,68.7 MHz): δ26.5 (C2′), 37.0 (C3′), 53.0 (OCH_(3ester)), 56.0(OCH_(3ether)), 66.7 (C1′), 108.3 (C3), 111.0 (C6), 121.6 (C_(arom)),140.9 (C2), 149.3 (C_(arom)), 152.6 (C_(arom)), 166.8 (C═O); MS (E/I)m/z (relative intensity): 284 (M^(+.), 90), 237 (70), 227 (93), 196(47), 181 (38), 137 (100), 122 (81), 93 (52), 79 (44); HRMS (E/I) exactmass calcd for C₁₂H₁₇N₂O₆: m/e 284.1008, obsd m/e 284.1018; IR(Nujol^(ò)) n: (cm⁻¹) 3472, 2937, 2911, 2855, 1733, 1532, 1516, 1462,1377, 1292, 1224, 1143, 1052, 884, 812, 792, 773, 756, 724, 646.

Synthesis of Methyl4-(N-9-fluorenylmethoxycarbonyl)aminopropyloxy-5-methoxy-2-nitrobenzoicacid (200)

A solution of 199 (3.9 g, 11.2 mmol) and KOH (1.9 g, 33.4 mmol) inaqueous methanol (77 mL, MeOH; 15 mL, H₂O) was heated at reflux for 90minutes. At which time TLC (EtOAc/MeOH/TEA 100:10:1) revealed completeconsumption of starting material. Excess MeOH was removed by evaporationunder reduced pressure and the concentrate diluted with H₂O (20 mL). Theaqueous solution was neutralised with conc. HCl, diluted with THF (100mL) and sodium carbonate (2.9 g, 27.9 mmol) was added to adjust thesolution to pH9. Fluorenylmethyl chloroformate (3.0 g, 11.6 mmol) wasadded portionwise over 30 minutes to the basic solution and the reactionmixture was allowed to stir for 12 hours. Excess THF was removed byevaporation under reduced pressure and the aqueous fraction wasextracted with EtOAc (3×100 mL) to remove excess fluorenylmethylchloroformate and related by-products. The aqueous layer was thenacidified with conc. HCl and extracted again with EtOAc (3×100 mL). Theorganic phase was washed with H₂O (2×100 mL), brine (100 mL), dried overMgSO₄, and excess solvent was removed by evaporation under reducedpressure to afford 200 as a beige solid which was not purified further,but used directly in the subsequent reaction (4.7 g, 86%). mp=145-146°C.; ¹H-NMR (CDCl₃, 270 MHz): δ1.81 (m, 2H, H2′), 3.43 (m, 2H, H3′), 3.78(s, 3H, OCH₃), 4.08-4.23 (m, 3H, H1′+Fmoc CH), 4.49 (d, 2H, J=6.41, FmocCH₂), 5.70 (br, 1H, NH), 7.14 (s, 1H, H6), 7.26-7.41 (m, 5H,Fmoc_(aryl)+H3), 7.59 (d, 2H, J=7.51 Hz, Fmoc_(aryl)), 7.74 (d, 2H,J=7.15 Hz, Fmoc_(aryl)), 9.62 (s, 1H, CO₂H); ¹³C-NMR (CDCl₃, 68.7 MHz):δ28.8 (C2′), 39.1 (C3′), 47.2 (CH Fmoc), 56.4 (OCH₃), 66.3 (CH₂Fmoc),68.5 (C1′), 107.9 (C3), 111.1 (C6), 120.0, 124.9, 127.1 and 127.7 (CHFmoc_(aryl)), 128.0 (C_(arom)), 137.0 (C_(arom)), 141.3 (C Fmoc_(aryl)),143.8 (C Fmoc_(aryl)), 148.2 (C_(arom)), 154.7 (C_(arom)), 156.8 (NC═O)171.5 (CO₂H); MS (FAB) m/z (relative intensity): 493 (M^(+.) +1, 3), 297(6), 271 (4), 191 (18), 180 (21), 179 (100), 178 (67), 165 (30), 102(17), 93 (13); HRMS (FAB) exact mass calcd for C₂₆H₂₅N₂O₈ (M+H): m/e493.1532, obsd m/e 493.1536; IR (Nujol^(ò)) n: (cm⁻¹) 1712, 1535, 1463,1377, 1277, 1219, 1081, 970, 762, 722, 656.

Synthesis of(2S)-N-[4-(N-9-fluorenylmethoxycarbonyl)aminopropyloxy-5-methoxy-2-nitrobenzoyl)]pyrrolidine-2-methanol(201)

A catalytic amount of DMF (2 drops) was added to a solution of thenitrobenzoic acid 200 (8.0 g, 16.3 mmol) and oxalyl chloride (2.3 g,17.9 mmol) in anhydrous DCM (120 mL), at room temperature under anitrogen atmosphere. The reaction mixture was stirred for 16 hours andthe resulting solution of acid chloride was cooled to 0° C.(ice/acetone) under a nitrogen atmosphere. A solution ofpyrrolidinemethanol (1.8 g, 17.9 mmol) and DIPEA (4.6 g, 35.77 mmol) inanhydrous DCM (40 mL) was added dropwise over 30 minutes. Once theaddition was complete, the reaction mixture was allowed to warm to roomtemperature. Stirring was continued for a further 2 hours, at which timeTLC (95% EtOAc/MeOH) revealed complete reaction. The reaction mixturewas washed with 1 N aqueous HCl (2×100 mL), H₂O (2×100 mL), brine (100mL), and dried over MgSO₄. Excess solvent was removed by evaporationunder reduced pressure to afford the crude compound as a brown oil.Purification by flash column chromatography (99% CHCl₃/MeOH) afforded201 as a beige solid (5.6 g, 82%). [α]²⁰ _(D)=−53.3° (c=1.03, CHCl₃);mp=78-81° C.; ¹H-NMR (CDCl₃, 270 MHz): δ1.69-1.88 (m, 4H, H4+H3),2.04-2.12 (m, 2H, H2′), 3.16 (m, 2H, H3′), 3.45 (m, 2H, H5), 3.81 (s,3H, OCH₃), 3.86-3.91 (m, 2H, CH₂—OH), 4.08-4.24 (m, 3H, H1′+Fmoc CH),4.38-4.48 (m, 3H, H2+Fmoc CH₂), 5.65 (br, 1H, NH), 6.78 (s, 1H,H6_(arom)), 7.27-7.42 (m, 5H, H3_(arom)+Fmoc_(aryl)), 7.61 (d, 2H,J=7.32 Hz, Fmoc_(aryl)), 7.76 (d, 2H, J=7.32 Hz, Fmoc_(aryl)); ¹³C-NMR(CDCl₃, 68.7 MHz): δ24.4 (C4), 28.4 (C3), 28.9 (C2′), 39.1 (C3′), 47.3(CH Fmoc), 49.5 (C5), 56.6 (OCH₃), 60.4 (C2), 61.5 (CH₂—OH), 66.2 (CH₂Fmoc), 68.5 (C1′), 108.0 (C3_(arom)), 108.9 (C6_(arom)), 120.0, 124.9,127.0 and 127.7 (CH Fmoc_(aryl)), 128.0 (C_(arom)), 137.0 (C_(arom)),141.3 (C Fmoc_(aryl)), 143.9 (C Fmoc_(ary)l), 148.2 (C_(arom)), 154.7(C_(arom)), 156.5 (NC═O_(carbamate)) 171.2 (C═O_(amide)); MS (FAB) m/z(relative intensity): 576 (M^(+.) +1, 32), 191 (18), 179 (100), 165(25), 102 (33); HRMS (FAB) exact mass calcd for C₃₁H₃₄N₃O (M+H): m/e576.2268 obsd m/e 576.2257; IR (Nujol^(ò)) n: (cm⁻¹) 2626, 1714, 1615,1576, 1520, 1452, 1434, 1333, 1276, 1218, 1147, 1059, 869, 818, 759,742.

Synthesis of (2S)-N-[4-(N-9-fluorenylmethoxycarbonyl)aminopropyloxy-5-methoxy-2-aminobenzoyl]pyrrolidine-2-methanol (202)

A mixture of the nitro compound 201 (5.5 g, 9.5 mmol) and SnCl₂/2H₂O(10.2 g, 45.4 mmol) in MeOH (100 mL) was heated at reflux and theprogress of the reaction monitoring by TLC (95% CHCl₃/MeOH). After 2hours excess MeOH was removed by evaporation under reduced pressure, theresulting residue was cooled (ice), and treated carefully with sat.aqueous NaHCO₃ (170 mL). The reaction mixture was diluted with EtOAc(170 mL) and after 16 hours stirring at room temperature the inorganicprecipitate was removed by filtration through Celite. The organic layerwas separated, washed with brine (150 mL), dried over MgSO₄, filteredand evaporated in vacuo to give a brown solid. Purification by flashcolumn chromatography (95% CHCl₃/MeOH) afforded the pure amine 202 as agreyish-pink solid (4.3 g, 82%). [α]²⁰ _(D)=−78.6° (c=1.02, CHCl₃); mp=83-86° C.; ¹-NMR (CDC₃, 270 MHz): δ1.68-1.85 (m, 4H, H4+H3), 2.00-2.04(m, 2H, H2′), 3.43-3.45 (m, 2H, H3′), 3.49-3.58 (m, 2H, H5), 3.67 (s,3H, OCH₃), 3.72-3.78 (m, 2H, CH₂—OH), 4.04 (t, 2H, J=5.58 Hz, H1′), 4.22(t, 1H, J=6.86 Hz, Fmoc CH), 4.41-4.44 (m, 3H, H2+Fmoc CH₂), 5.92 (br,1H, NH), 6.23 (s, 1H, H3_(arom)), 6.71 (s, 1H, H6_(arom)), 7.27-7.41 (m,4H, Fmoc_(aryl)), 7.62 (d, 2H, J=7.32 Hz, Fmoc_(aryl)), 7.75 (d, 2H,J=7.33 Hz, Fmoc_(aryl)); ¹³C-NMR (CDCl₃, 68.7 MHz): δ24.9 (C4), 28.6(C3), 29.1 (C2′), 39.5 (C3′), 47.3 (CH Fmoc), 51.0 (C5), 56.6 (OCH₃),60.4 (C2), 61.1 (CH₂—OH), 66.4 (CH₂ Fmoc), 68.0 (C1′), 102.0(C3_(arom)), 111.6 (C6_(arom)), 120.0, 125.1, 127.0 and 127.7 (CHFmoc_(aryl)), 128.0 (C_(arom)), 137.8 (C_(arom)), 141.3 (C Fmoc_(aryl)),144.0 (C Fmoc_(aryl)), 148.2 (C_(arom)), 150.8 (C_(arom)), 156.6(NC═O_(carbamate)), 171.9 (C═O_(amide)); MS (FAB) m/z (relativeintensity): 546 (M^(+. +)1, 11), 445 (10), 191 (14), 179 (100), 166(51), 102 (70); HRMS (FAB) exact mass calcd for C₃₁H₃₇N₃O₆ (M+H); m/e546.2526 obsd m/e 546.2532; IR (Nujol^(ò)) n: (cm⁻¹) 1698, 1622, 1588,1506, 1476, 1404, 1228, 1173.

Synthesis of(2S)-N-[4-(N-9-fluorenylmethoxycarbonyl)aminopropyloxy-5-methoxy-2-(N-2,2,2-trichloroethyloxycarbonyl)aminobenzoyl]pyrrolidine-2-methanol(203)

A solution of the amine 202 (1.1 g, 2.0 mmol) in DCM (40 mL) was cooledto 0° C. (ice/acetone bath) and treated with pyridine (0.33 mL, 0.3 g,4.1 mmol). A solution of trichloroethyl chloroformate (0.27 mL, 0.41 g,1.9 mmol) in DCM (10 mL) was added dropwise over 30 minutes to thestirred mixture. The reaction mixture was allowed to warm to roomtemperature and stirred for a further 3 hours, at which time TLC (EtOAc)revealed complete loss of starting material. The reaction mixture waswashed with 1 N HCl solution (50 mL), H₂O (2×50 mL), brine (50 mL),dried over MgSO₄, filtered and evaporated in vacuo. The crude residuewas purified by flash column chromatography (98% CHCl₃/MeOH) to affordthe pure trichloroethyl-carbamate 203 as a brown solid (1.1 g, 74%).[α]²⁰ _(D)=−35.7° (c=0.87, CHCl₃); mp=54-57° C.; ¹H-NMR (CDCl₃, 270MHz): δ1.73-1.89 (m, 2H, H4), 2.00-2.04 (m, 2H, H2′), 2.18 (m, 2H, H3),3.44-3.54 (m, 4H, H3′+H5), 3.72 (s, 3H, OCH₃), 3.81-3.90 (m, 2H, CH213OH), 4.14-4.25 (m, 3H, H1′+Fmoc CH), 4.43-4.45 (m, 3H, Fmoc CH₂+H2),4.76 (d, 1H, J=12.00 Hz, Troc CH₂), 4.83 (d, 1H, J=12.00 Hz, Troc CH₂),5.89 (br, 1H, Fmoc NH), 6.82 (s, 1H, H6_(arom)), 7.26-7.41 (m, 4H,Fmoc_(aryl)), 7.62 (d, 2H, J=7.33 Hz, Fmoc_(aryl)), 7.69 (s, 1H,H3_(arom)), 7.75 (d, 2H, J=7.51 Hz, Fmoc_(aryl)), 9.06 (br s, 1H, TrocNH); ¹³C-NMR (CDCl₃, 68.7 MHz): δ25.0 (C4), 28.2 (C3), 28.9 (C2′), 39.5(C3′), 47.3 (CH Fmoc), 51.4 (C5), 56.1 (OCH₃), 60.8 (C2), 66.0 (CH₂—OH),66.3 (CH₂ Fmoc), 68.2 (C1′), 74.4 (CH₂ Troc), 95.3 (C_(quat)), 105.6(C3_(arom)), 110.7 (C6_(arom)), 120.0, 125.1, 127.0 and 127.7(C—H_(aryl) Fmoc), 130.7 (C_(arom)), 141.3 (C_(aryl) Fmoc), 144.0(C_(aryl) Fmoc), 144.5 (C_(arom)), 150.0 (C_(arom)), 152.1(NC═O_(carbamate) Troc), 156.5 (NC═O_(carbamate) Fmoc), 170.4(NC═O_(amide)); MS (FAB) m/z (relative intensity): 720 (M^(+.) +1, 2),275 (4), 192 (29), 179 (100), 166 (13), 102 (48), 70 (10); IR(Nujol^(ò)) n: (cm⁻¹) 3338, 1742, 1714, 1599, 1520, 1464, 1378, 1215,1170, 1119, 1024, 817, 759, 740.

Synthesis of(11S,11aS)-10-N-2,2,2-trichloroethyloxycarbonyl-11-hydroxy-8-(N-9-fluorenylmethoxycarbonyl)aminopropyloxy-7-methoxy-1,2,3,6,9,11a-hexahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(204)

All glassware, needles and cannulae used for this procedure had beenpreviously dried overnight in an oven, and were assembled while stillwarm and the enclosed vessel flooded with nitrogen and evacuated threetimes. Freshly distilled DCM (6.6 mL) was transferred to the reactionvessel and the temperature lowered to −45° C. (dry ice/CH₃CN) under anitrogen atmosphere. Oxalyl chloride (1.0 mL of a 2 M solution in DCM,2.0 mmol) was transferred to the reaction vessel, followed by thedropwise addition over 30 minutes of anhydrous DMSO (0.3 mL, 0.3 g, 3.9mmol) in dry DCM (4.2 mL). After stirring at −45° C. for 30 minutes, asolution of the alcohol 203 (0.79 g, 1.1 mmol) dissolved in dry DCM (6.6mL) was added dropwise over 50 minutes. The reaction mixture was allowedto stir at −45° C. for 45 minutes, the mixture was then treated dropwisewith DIPEA (1.9 mL, 1.4 g, 10.8 mmol) in dry DCM (4.2 mL) over 30minutes at −45° C. After 35 minutes, TLC (97% CHCl₃/MeOH) revealedcomplete consumption of starting material. The reaction mixture wasallowed to warm to room temperature, diluted with DCM (30 mL), washedwith 1 N HCl solution (30 mL), H₂O (30 mL), brine (40 mL), dried overMgSO₄, filtered and evaporated in vacuo. Purification by flash columnchromatography (97% CHCl₃/MeOH) furnished the protected carbinolamine204 as a brown solid (0.48 g, 78%). [α]²⁰ _(D)=+62.3° (c=0.83, CHCl₃);mp=76-79° C.; ¹H-NMR (CDCl₃, 270 MHz) δ2.00-2.17 (m, 6H, H2+H2′+H1),3.43-3.60 (m, 3H, H3′+H11a), 3.66-3.73 (m, 2H, H3), 3.78 (s, 3H, OCH₃),4.20-4.32 (m, 4H, H1′+Fmoc CH+1H Troc CH₂), 4.44 (d, 2H, J=6.78 Hz, FmocCH₂), 5.25 (d, 1H, J=12.00 Hz, Troc CH₂), 5.65 (d, 1H, J=9.71 Hz, H11),5.87 (br, 1H, NH), 6.82 (s, 1H, H6), 7.23-7.41 (m, 5H, H9+Fmoc_(aryl)),7.61 (d, 2H, J=7.32 Hz, Fmoc_(aryl)), 7.75 (d, 2H, J=7.51 Hz,Fmoc_(aryl)); ¹³C-NMR (CDCl₃, 68.7 MHz) δ23.0 (C2), 28.6 (C1), 29.0(C2′), 39.5 (C3, ), 46.4 (C3), 47.3 (CH Fmoc), 56.0 (OCH₃), 60.0 (C11a),66.4 (CH₂ Fmoc), 68.3 (C1′), 74.9 (CH₂ Troc), 86.4 (C1), 95.1 (C_(quat))110.5 (C16), 113.8 (C9), 120.0, 125.1, 127.0 and 127.7 (C-H_(aryl)Fmoc), 128.8 (C_(arom)) 130.9 (C_(arom)), 141.3 (C_(aryl) Fmoc), 143.9(C_(aryl) Fmoc), 148.8 (C_(arom)), 149.9 (C_(arom)), 154.4(NC═O_(carbamate) Troc), 156.6 (NC═O_(carbamate) Fmoc), 167.0(C4_(amide)); MS (FAB) m/z (relative intensity): 702 (6), 275 (3), 192(16), 179 (100), 165 (18), 102 (21), 70 (15); IR (Nujol^(ò)) n: (cm⁻¹)3383, 2970, 2946, 2880, 2844, 1713, 1602, 1513, 1464, 1377, 1218, 1034,908, 723, 645.

Synthesis of (11aS)8-(N-9-fluorenylmethoxycarbonyl)aminopropyloxy-7-methoxy-1,2,3,11a-tetrahydro-5H-pyrrolo[2,1-c][1,4]benzodiazepin-5-one(205)

Yellow lead (II) oxide (500 mg, 2.24 mmol) was dissolved in 50% aqueousacetic acid (5 mL) and the solution added slowly to a vigorously stirredsuspension of cadmium dust (2.5 g, 22.4 mmol) in de-ionised H₂O (10 mL).The cadmium darkened as lead deposited on the surface and the clumpswere broken up carefully. After 20 minutes, the solid couple wasfiltered under vacuum, washed with H₂O and acetone and dried in vacuo.The lumps were crushed and stored in a closed vial.

The cadmium/lead couple (0.62 g, equiv. 0.56 g, 4.94 mmol Cd) was addedin one portion to a solution of the Troc protected carbinolamine 204(0.71 g, 0.99 mmol) and ammonium acetate (1.0 M, 9 mL) in THF (9 mL) atroom temperature. The reaction mixture was stirred for 4 hours, duringwhich time the reaction mixture became cloudy and opaque with a fluffywhite precipitate. When reaction was complete as indicated by TLC (95%CHCl₃/MeOH), the solids were removed by filtration through Celite, andthe THF removed by evaporation under reduced pressure. The filter cakewas washed with several aliquots of EtOAc. The aqueous layer wasextracted with EtOAc (3×15 mL), and the organic phase was dried overMgSO₄, filtered and evaporated in vacuo. Purification by flash columnchromatography (97% CHCl₃/MeOH) furnished the target compound 205 as abrown solid (0.47 g, 90%) which was repeatedly evaporated in vacuo withCHCl₃ in order to obtain the N10—C11 imine form of the compound. [α]²⁰_(D)+385.1° (c=0.47, CHCl₃); mp=73-76° C; ¹H-NMR (CDCl₃, 270 MHz):δ2.04-2.06 (m, 4H, H2 +H1), 2.27-2.29 (m, 2H, H2′), 3.45-3.47 (m, 2H,H3′), 3.67-3.73 (m, 2H, H3), 3.80 (s, 3H, OCH₃), ), 3.84-4.23 (m, 4H,H11a+H1′+Fmoc CH), 4.43-4.46 (m, 2H, Fmoc CH₂), 5.92 (br, 1H, NH), 6.82(s, 1H, H6), 7.29-7.41 (m, 4H, Fmoc_(aryl)), 7.5 (s, 1H, H9), 7.61 (d,2H, J=7.14 Hz, Fmoc_(aryl)), 7.67 (d, 1H, J=4.40 Hz, H11_(imine)), 7.75(d, 2H, J=7.33 Hz, Fmoc_(aryl)); ¹³C-NMR (CDCl₃, 68.7 MHz): δ22.3 (C2),29.3 (C1), 29.6 (C2′), 39.6 (C3′), 46.7 (C3), 47.4 (CH Fmoc), 53.7(OCH₃), 56.0 (C11a), 66.3 (CH₂ Fmoc), 68.3 (C1′), 110.2 (C6), 111.4(C9), 120.0 (C—H_(aryl) Fmoc), 120.5 (C_(arom)), 125.1, 127.0, and 127.7(C—H_(aryl) Fmoc), 140.6 (C_(arom)), 141.3 (C_(aryl) Fmoc), 144.0(C_(aryl) Fmoc), 147.7 (C_(arom)), 150.3 (C_(arom)), 156.6(NC═O_(carbamate)), 162.5 (C11), 164.5 (C4_(amide)); MS (FAB) m/z(relative intensity): 526 (M^(+.) 1, 15), 348 (7), 330 (4), 304 (4), 247(12), 191 (15), 179 (100), 165 (17), 102 (40), 91 (10), 70 (13); HRMS(FAB) exact mass calcd for C₃₁H₃₂N₃O, (M+H): m/e 526.2264 obsd m/e526.2198; IR (Nujol^(ò)) n: (cm⁻¹) 3327, 1729, 1690, 1601, 1509, 1427,1261, 1217, 1023, 759, 740, 699.

Examples 5 to 8

Cytotoxicity Data

NCI In vitro Cytotoxicity Studies

The National Cancer Institute (NCI), Bethesda, Md., USA has available anin vitro cytotoxicity screen which consists of approximately 60 humantumour cell lines against which compounds are tested at a minimum offive concentrations each differing 10-fold. A 48 hour continuousexposure protocol is used, where cell viability or growth is estimatedwith an SRB protein assay.

Method

The test compounds were evaluated against approximately 60 human tumourcell lines. The NCI screening procedures were described in detail byMonks and co-workers (Monks, A et al., Journal of the National CancerInstitute, 1991, 83, 757). Briefly, cell suspensions were dilutedaccording to the particular cell type and the expected target celldensity (5000-40,000 cells per well based on cell growthcharacteristics), and added by pipette (100 μL) into 96-well microtitreplates. The cells were allowed a preincubation period of 24 hours at 37°C. for stabilisation. Dilutions at twice the intended test concentrationwere added at time zero in 100 μL aliquots to the wells. The testcompounds were evaluated at five 10-fold dilutions (10⁻⁴, 10⁻⁵, 10⁻⁶,10⁻⁷ and 10⁻⁸ μM). The test compounds were incubated for 48 hours in 5%CO₂ atmosphere and 100% humidity. The cells were then assayed using thesulphorhodamine B assay. A plate reader was used to read the opticaldensities and a microcomputer processed the readings into LC₅₀ values,which is the dosage required to kill half of the cells.

The results presented in examples 5 to 8 are LC₅₀values which are below10 μM, which is taken to be the dividing line between cytotoxicity andnon-cytotoxicity.

NCI Hollow Fibre Assay for Preliminary in vivo Testing

The Biological testing Branch of the Developmental Therapeutics Programof the NCI has adopted a preliminary in vivo screening tool forassessing the potential anticancer activity of compounds identified bythe large scale in vitro cell screen. For these assays, human tumourcells are cultivated in polyvinylidene (PVDF) hollow fibres, and asample of each cell line is implanted into each of two physiologiccompartments (intraperitoneal and subcutaneaous) in mice. Each testmouse received a total of 6 fibres (3 intraperitoneally and 3subcutaneously) representing 3 distinct cancer cell lines. These miceare treated with potential antitumour compounds at each of 2 test dosesby the intraperitoneal route using a QD×4 treatment schedule. Vehiclecontrols consist of 6 mice receiving the compound diluent only. Thefibre cultures are collected on the day following the last day oftreatment. To assess anticancer effects, the viable cell mass isdetermined for each of the cell lines using a formazyn dye (MTT)conversion assay. From this, the % T/C can be calculated using theaverage optical density of compound treated samples divided by theaverage optical; density of the vehicle controls. In addition, the netincrease in cell mass can be determined for each sample, as a sample offibre cultures are assessed for viable cell mass on the day ofimplantation into mice. Thus, the cytostatic and cytocidal capacities ofthe test compound can be assessed.

Generally, each compound is tested against a minimum of 12 human cancercell lines. This represents a total of 4 experiments since eachexperiment contains 3 cell lines. The data are reported as % T/C foreach of the 2 compound doses against each of the cell lines withseparate values calculated for the intraperitoneal and subcutaneoussamples.

Compounds are selected for further in vivo testing in standardsubcutaneous xenograft models on the basis of several hollow fibre assaycriteria. These include: (1) a % T/C of 50 or less in 10 of the 48possible test combinations (12 cell lines×2 sites×2 compound doses); (2)activity at a distance (intraperitoneal drug/subcutaneous culture) in aminimum of 4 of the 24 possible combinations; and/or (3) a net cell killof 1 or more of the cell lines in either implant site. To simplifyevaluation, a points system has been adopted which allows rapidevaluation of the activity of a given compound. For this, a value of 2is assigned for each compound dose which results in a 50% or greaterreduction in viable cell mass. The intraperitoneal and subcutaneoussamples are scored separately so that criteria (1) and (2) can beevaluated. Compounds with a combined IP+SC score of 20, a SC score of 8or a net cell kill of one or more cell lines are referred for xenografttesting. This comparison indicated that there was a very low probabilityof missing an active compound f the hollow fibre assay was used as theinitial in vivo screening tool. In addition to these criteria, otherfactors (e.g. unique structure, mechanism of action) may result inreferral of a compound for xenograft testing without the compoundmeeting these criteria.

NCI Human Xenograft Studies

These are carried out on nude athymic mice with a disabled immunesystem. The human tumour tissue to be tested is implanted in theirflanks, and whilst the control mouse receives no treatment, the othersare subjected to varying doses of the test compound, which isadministered intraperitoneally. The results are expressed as thetoxicity of the compound, the amount of tumour growth, and theinhibition of growth.

Example 5

In vitro Cytotoxicity of Compounds of Formula I

Some of the compounds synthesised in example 1, were subjected to theNCI In Vitro Cytotoxicity study. The results (LC₅₀; μM) are set outbelow, and are illustrated in FIG. 28.

TUMOUR CELL-LINE UP2003 UP2051 UP2052 UP2065 TYPE DESIGNATION (24) (31)(33) (42) LC₅₀(μM) Lung NCI-H23 9.3 NCI-H460 7.6 3.0 NCI-H522 3.1 ColonCOLO 205 1.4 4.0 HCC-2998 5.2 5.2 0.8 HCT-116 1.1 KM12 9.5 CNS SNB-756.0 Melanoma MALME-3M 0.7 5.1 4.7 M14 2.7 SK-MEL-2 7.6 0.5 3.5 UACC-620.7 Renal 786-0 3.0 RXF 393 0.8 0.8 Breast MDA-MB-435 0.8Of the compounds tested, the above showed cytotoxicity against humanlung, colon, CNS, melanoma, renal and breast cancer cell lines.Replacing the C-8 benzyloxy group in UP2003 (24) with a methoxysubstituent (UP2065, 42) significantly changed the cytoxicity profile,activity was lost against lung, CNS, and colon cancer cell lines (onlyreduced activity against Colo 205 remained). However, additionalcytotoxic activity was gained against the melanoma cell lines SKMEL-2and MALME-3M, the renal cell line RXF-393 and the breast cell lineMDA-MB-435. Reduction of the ester moiety in UP2003 (24) to afford thealcohol UP2052 (33) resulted in increased activity in the lung cancercell line NCI-460 and the colon cell line HCC-2998. Additional activitywas registered against the lung cell line NC1—H522, the colon cell lineHCT-116, the melanoma cell line M14 and the renal cancer cell line786-0. Interestingly, the acetylated analogue UP2051 (31) exhibitedattenuated or abolished activity in these cell lines (e.g. 7.6 μM verses0.5 μM for UP2052 in the melanoma SK-MEL-2 cell line.

Example 6(a)

In vitro Cytotoxicity of Compounds of Formula II

Some of the compounds synthesised in example 2, were subjected to theNCI In Vitro Cytotoxicity study. The results (LC₅₀; μM) are set outbelow, and are illustrated in FIG. 29.

CELL-LINE TUMOUR DESIG- UP2064 UP2001 UP2004 UP2023 UP2067 TYPE NATION(74) (80) (70) (64) (172) LC₅₀(μM) Lung NCI-H23 7.6 NCI-H226 9.1NCI-H460 2.7 NCI-H522 5.2 5.0 Colon COLO 205 0.6 3.9 5.8 5.8 HCC-29980.099 5.5 7.0 KM12 7.1 CNS SF-539 9.4 6.8 SNB-75 7.5 5.4 Melanoma MALME-0.9 0.073 7.8 7.4 3M M14 0.8 SK-MEL-2 1.7 7.4 SK-MEL-28 2.6 8.4 6.6SK-MEL-5 7.8 6.0 UACC-257 7.4 7.3 UACC-62 0.6 0.077 5.3 7.2 3.0 RenalRXF 393 0.8 6.1 0.8 Breast MDA-MB- 2.3 7.6 0.8 435 MDA-N 9.0 6.6 0.6Of the compounds tested, the above listed exert their cytotoxic effect(LC₅₀) most strongly in the Lung, Colon, CNS. Melanoma, Renal and Breastcell line panels. Within the group, it is apparent that exchanging a C-8benzyloxy substituent (UP2004, 70) for a methoxy group (UP2064, 74)results in increased activity in the Melanoma panel. The methoxyanalogue is more potent and acts against a greater number of cell lines.The methoxy analogue also exhibits improved activity against the coloncancer cell line Colo 205 and, in addition, the methoxy analogueexhibits activity against the renal cell line RXF-393 which is notobserved with the benzyloxy compound. Replacing the electron richdimethoxy A-ring with an iodo substituted aromatic ring (UP2023, 64)resulted in slight attenuation of activity in some cell lines, but theanalogue showed activity against a wider spread of cell lines (i.e. 5melanoma cell lines against only 3 for the benzyloxy analogue). Changingthe nature of the C-ring ex-unsaturation from an alkene to a ketone(UP2067, 172) lead to additional activity against the breast cancer cellline MDA-MB-435, renal cell line RXF-393, the melanomas MALME-3M, M14,SKMEL-28, the CNS cancers SF-539 and SNB-75 and against the lung cellline NCI-H522.

The PBD dimer UP2001 (80) exhibited potent and selective cytoxicityactivity against the lung cancer cell line NCI-H460, the colon cell lineHCC-2998, the CNS cancer cell line SNB-75 and the melanoma cell linesMALME-3M (very potent, 0.08 μM) and UACC-62 (very potent, 0.07 μM),which may be attributable to its ability to cross link DNA.

Example 6(b)

Hollow Fibre Assay on Compounds of Formula II

Two of the compounds tested underwent the NCI Hollow Fibre Assay, andthe results are presented below.

UP2001 (80) UP2004 (70) IP score 40  8 SC score 14 10 Total score 54 18Cell Kill Y NUP2001 (80) and UP2004 (70) were subjected to the NCI Hollow Fibre assaydescribed above. UP2001 has been selected for xenograft studies based onits combined IP+SC score (54) which was greatly in excess of 20, and itsSC score which was higher than 8. UP2004 has been selected on the basisof its SC score, it being higher than 8.

Example 6(c)

Human Xenograft Studies on Compound 80 (UP 2001)

Human tumour xenograft studies on UP2001 were performed by theBiological Testing Branch of the NCI as described above.

Athymic nude mice bearing MDA-MB-435 xenografts (human mammary tumour),Ovcar-3 (human ovarian tumour), UACC-62 (human melanoma) or OVCAR-5(human ovarian tumour) were treated at doses of 0.67 (high), 0.45(middle) and 0.3 (low) mg/kg/injection given once every 4th day for atotal of 3 doses (6 mice per dose level with 20 controls).

UP2001 (80) was evaluated by measuring the toxicity of the drug and itsability to retard tumour growth.

Toxicity % T/C % Growth Delay Tumour High Mid Low High Mid Low High MidLow MDA- 3/6 1/6 2/6 toxic  3  3 41 41 41 MB-435 OV- 0/6 0/6 0/6  7 2046 73 73  9 CAR-3 UACC- 0/6 0/6 0/6 22 28 67 43 43 43 62 OV- 0 0/6 0/652 45 38 16 28 32 CAR-5Toxicity represents the number of mice which died as a result oftreatment. % T/C represents the width of the tumours in the “test” mice(T) (as measured with calipers) compared to control untreated mice (C)and presented as a percentage. % Growth Delay represents the increase inthe amount of time taken for the tumors to reach an arbitrary size of250 mg.

In the MDA-MB-435 xenografts UP2001 restricted tumour growth in treatedmice to only 3% of the tumour growth observed in the control population.In addition, a 41% delay in the time taken to reach tumour mass of 250mg was also observed. Some toxicity towards the hosts was observed evenat low dose.

A good dose response was observed for UP2001 (80) in the Ovcar-3xenografts. At the high dose, tumour growth in treated subjects was only7% of that observed in the control population. At the medium dose thevalue was 20% and at the low dose the tumours in the treated mice were46% of the size of the control tumours.

At the high dose a 73% growth delay in reaching a tumour mass of 250 mgwas observed. No mice died as a result of exposure to UP2001 (80).

A similar dose response for tumour growth was observed in the UACC-62xenografts for UP2001 (80). At the high dose treated tumours were 22% ofthe size of the control tumours. At the medium dose treated tumours were28% of the size of the control tumours and at the low dose treatedtumours were 67% of the size of the control tumours. Again no mice diedas a result of exposure to UP2001 (80).

Results for the human ovrian tumour OVAR-5 were less clear cut;approximately 50% tumour size reduction was observed and growth delaywas observed but activity appeared to be higher at lower concentrations.However, again, no mice died as a result of exposure to UP2001 (80).

UP2001 (80) was also evaluated against the human CNS tumour SF-295.Athymic nude mice bearing SF-295 were treated at doses of 0.40, 0.27 and0.18 mg/Kg by injection given intravenously once daily for a total of 5doses.

Toxicity % T/C Tumour Free High Med Low High Med Low High Med Low 2/61/6 2/6 0% 0% 0% 4/4 5/5 3/4UP2001 (80) displayed curative properties against SF-295 xenografts. Athigh and medium doses all the surviving mice were tumour free on day 27of the experiment. At the lower dose 3 out of 4 mice were tumour free onday 27. Some toxicity was associated with the treatment, 2 mice dying atthe high dose, 1 at the medium dose and two at the low dose. The higherintensities of the injection schedule may be reflected in the highermortality observed.

Example 7

In vitro Cytotoxicity of Compounds of Formula III

All of the compounds synthesised in example 3, were subjected to the NCIIn Vitro Cytotoxicity screen. The results (LC50;μM) are set out below,and are illustrated in FIG. 30.

TUMOUR CELL-LINE UP2026 UP2027 UP2028 UP2068 TYPE DESIGNATION (136)(138) (151) (96) LC₅₀(μM) Lung NCI-H522 7.8 8.0 0.8 8.5 Colon COLO 2058.8 5.0 HCC-2998 6.4 KM12 8.8 CNS SNB-75 8.2 Melanoma MALME-3M 6.1 5.78.3 LOX IMVI 9.7 M14 7.8 6.5 SK-MEL-2 7.4 9.5 5.4 8.1 SK-MEL-28 7.1 8.19.6 SK-MEL-5 9.0 UACC-257 7.7 UACC-62 6.6 Renal RXF 393 7.6 6.6 0.7 6.3Breast HS 578T 9.2 MDA-MB-435 6.3 7.2 8.3 MDA-N 6.3The C-7-phenyl substituted compound UP2026 (136) showed cytotoxicityagainst cell lines in the human lung, colon, melanoma, renal and breastcancer panels. Interestingly, unlike other PBDs the molecule wasinactive in the CNS cell line panel. However, UP2026 (136) was activeagainst nearly all the members of the melanoma panel. Inclusion of amethoxy group in the C7 aryl moiety (138) resulted in increasedselectivity as cytoxicity was only observed in the lung cell lineNCI-H522, the melanoma cell line SKMEL-2 and the renal cell lineRXF-393. Introduction of a nitro group at C7 completely abolishedcytotoxic activity, however, it seems likely that activity would berestored once the nitro group is reduced to an amine; in this way UP2029(140) might prove to be a useful prodrug with potential use in treatinglarge hypoxic tumours. The C8 amino substituted PBD (UP2028, 151) showedgood activity in the lung, colon, CNS, melanoma, renal and breast cellline panels. On the other hand the trimethoxy PBD (UP2068, 96) was onlyactive in the lung, melanoma, renal and breast cell line panels.

Example 7

In vitro Cytotoxicity of Compounds of Formula III

All of the compounds synthesised in example 3, were subjected to the NCIIn Vitro Cytotoxicity screen. The results (LC50; μM)are set out below,and are illustrated in FIG. 30.

TUMOUR CELL-LINE UP2005 UP2008 TYPE DESIGNATION (161) (167) LC₅₀(μM)Lung NCI-H23 8.9 NCI-H522 8.7 Colon HCC-2998 8.1 CNS SF-295 8.8 SF-5397.7 Melanoma MALME-3M 7.5 6.8 LOX IMVI 9.2 M14 6.2 8.4 SK-MEL-2 7.6 6.5SK-MEL-28 6.5 UACC-257 7.1 Renal RXF 393 6.8Two of the four C8 PBD amides, UP2005 (161) and UP2008 (167),demonstrated cytotoxicity (LC₅₀) in the NCI assay. UP2005 (161) showedselectivity for the lung, CNS, melanoma and renal cancer in panels. Thecompound was particularly active in the melanoma panel exhibitingcytotoxicity against 5 out of the 8 melanoma cell lines. UP2008 (167)revealed a slightly different profile being active in the lung, colon,and melanoma panels. Again the molecule was particularly active in themelanoma panel.

Example 9

Further Results for PBD dimer SJG-136 (UP2001, 80)

The compound synthesized in example 2(d) (SJG-136, 80) underwent somefurther assays.

The first assay, which is described in G. B. Jones, et al., Anti-CancerDrug Des., 1990, 5, 249, which is incorporated herein by reference,determines the effect of the test compound on the helix meltingtemperature of DNA. This assay is designed to give an indication of thestrength and extent of cross-linking of the DNA strands by the testcompound (i.e. a measure of the stabilisation of the DNA upon ligandbinding).

The melting temperature was determined for a 1:5 molar ratio of [ligand][DNA], where the calf thymus DNA concentration is 100 mM in aqueoussodium phosphate buffer (10 mM sodium phosphate +1 mM EDTA,pH7.00±0.01). For calf thymus DNA at pH7.00±0.01, the meltingtemperature is 67.83±0.06° C. (mean value from 30 separatedeterminations).

For a 1:5 molar ratio of [PBD]:[DNA], the PBD dimer 80 elevates thehelix melting temperature (ΔT_(m)) of calf thymus DNA by anunprecedented 33.60C after incubation for 18 hours at 37° C. Underidentical conditions, the C-ring-unsubstituted dimer DSB-120:

provides a ΔT_(m) of 15.1° C., demonstrating the extraordinary effect ofintroducing C2/C2′-unsaturation. In common with other PBD dimers, 80exerts most of its effect upon the GC-rich or high temperature regionsof the DNA melting curves. In a similar fashion to DSB-120, it providessome 60-80% of its stabilising effect without prior incubation,suggesting a kinetic effect in the PBD reactivity profile. However, thecomparative ΔT_(m) curves show that, on a concentration basis alone,SJG-136 is ≧10-fold more effective than DSB-120. Even at a [PBD]:[DNA]molar ratio of 1:100, SJG-136 still exhibits significantly better DNAbinding affinity than the monomer tomaymycin at a 1:5 [PBD][DNA] molarratio.

The results for a [PBD]:[DNA] ratio of 1:5 are summarised in the tablebelow (All ΔTm values ±0.1-0.2° C.)

Induced ΔT_(m) (° C.) after incubation at 37° C. for Compound 0 h 4 h 18h SJG-136 (80) 25.7 31.9 33.6 DSB-120 10.2 13.1 15.1 Tomamycin 0.97 2.382.56The data presented in the above table show that SJG-136 (80) is the mostpotent DNA-stabilising agent known to date according to this particularassay.

The second assay determined the cytotoxicity of SJG-136 (80) in thehuman ovarian carcinoma cell line A2780 and its cisplatin-resistantsubline A2780cis R, and compared this data with the cytotoxicity of therelated dimer DSB-120 (see above) and Cisplatin. Relative to theparental line, the A2780cis R subline is known to have elevated GSHlevels, an increased level of repair of DNA-cisplatin adducts, and adecreased ability to uptake cisplatin (M. Smellie, et al., Br. J.Cancer, 1994, 70, 48).

The results, which were obtained by incubating the cells with thecompounds for 96 hours at 37° C., and assessing the cell number usingSulforhodamine B, are presented in the table below:

IC₅₀ ^(a) (μM) for A2780 A2780cis^(R) RF^(b) SJG-136 (80) 0.0000230.000024 1.1 DSB-120 0.0072 0.21 29.2 Cisplatin 0.265 8.4 32 ^(a)Dose ofcompounds required to inhibit cell growth by 50% compared with control^(b)RF is the resistance factor (IC₅₀ resistant/parent)The IC₅₀ value for 80 in the A2780 cell line is only 23 pM, representinga 320-fold increase in cytotoxicity compared to DSB-120 (IC₅₀=7.2 nM).More interestingly, whereas DSB-120 has a reduced potency in thecisplatin-resistant A2780cis R (IC₅₀=0.21 mM), SJG-136 is almost9,000-fold more potent in this cell line with a similar IC₅₀ value (24pM) to that in the normal A2780, giving a Resistance Factor of 1.1. Thefact that both DSB-120 and cisplatin give Resistance Factors of 29.2 and32, respectively, across this pair of cell lines suggests that SJG-136may have potential in the treatment of cisplatin-refractory disease.

Example 10

Ovarian Carcinoma Cytotoxicity Assay

Compounds of the invention (and Anthramycin as a comparison) wereevaluated for their cytotoxic activity in ovarian cell lines by Dr LloydR. Kelland's group at The Institute of Cancer Research, Sutton, UK. Thefive cell lines investigated were SKOV-3, A2780/A2780cisR andCH1/CH1cisR (cisR denotes that the cell line is resistant to cisplatin).

Single viable cells were seeded in growth medium (160 L) in 96-wellmicrotitre plates and allowed to attach overnight. The PBDs were thendissolved in DMSO (to give 20 mM drug concentrations) immediately priorto adding to the cells in quadruplicate wells. The final drugconcentrations in the wells ranged from 100 μM to 2.5 nM as follows:100, 25, 10, 2.5, 1 μM, 250, 100, 25, 10, 2.5 nM (drugs were diluted ingrowth medium and then 40 μL added to the existing well volume of 160 μLto give final concentrations as above). After 96 hours, the medium wasremoved and the remaining cells fixed by exposure to 10% trichloroaceticacid on ice for 30 minutes. The wells were then washed 3-4 times withtap water, air dried overnight and treated with 100 μL ofsulphorhodamine B (0.4%) dissolved in 1% acetic acid. Staining wasallowed to continue for 10-15 minutes, then the wells were washed 3-4times with 1% acetic acid, air dried and then added to Tris base (100 μLof 10 mM). Plates were then shaken and absorbance readings at 540 nmwere determined using a plate reader. By using the Quattro-Pro softwarepackage, the IC₅₀ values were calculated from plots of concentrationversus percentage absorbance (compared with 8 untreated wells).

(a) Compounds of Formula I IC₅₀ (μM) Compound A2780 A2780cisR CH1CH1cisR Skov3 Anthramycin 0.155 0.16 0.062 0.05 0.16 UP2003 (24) 0.01450.12 0.016 0.04 0.012 UP2051 (31) 0.1 0.27 0.105 0.16 0.46 UP2052 (33)0.07 0.105 0.09 0.037 0.105 UP2053 (56) 0.0054 0.058 0.0115 0.011 0.1UP2065 (42) 0.36 0.46 0.115 0.15 0.45 UP2074 (10) 0.155 0.43 0.105 0.270.52 UP2089 (177) 0.0022 0.0042 <0.0025 0.0023 0.0054 UP2092 (179) 0.0040.007 0.0016 0.0082 0.0098 UP2095 (181) <0.05 <0.05 <0.05 <0.05 <0.05The most potent members of this group of compounds are those PBDs thatpossess aryl or vinyl substitution at the 2 position of the PBD: UP2089(177), UP2092 (179) and UP2095 (181). Without wishing to be bound bytheory, the potent activity of these molecules can probably be ascribedto the presence of conjugated endo-exo unsaturation in these molecules.Endo-exo unsaturation may improve the fit of the molecule in the minorgroove of DNA, although the conjugated system may also indirectly affectthe potency of the molecules through electronic and conformationaleffects. UP2089 (177) and UP2092 (181) are up to 100 times more potentthan the natural product anthramycin, which also possesses conjugatedendo exo unsaturation.

PBD dimers are able to cross-link DNA and block tumour cell replicationand thus generally show high cytotoxicity. The PBD dimer UP2053, whichpossesses only endo unsaturation, exhibits potent activity in theseovarian cell lines. The dimer is markedly more cytotoxic thananthramycin but not as potent as the monomers UP2089 and 2092.

The remaining molecules of Formula I are monomers possessing only endounsaturation, these molecules are broadly comparable with anthramycin.However, the ester UP2003 and the alcohol UP2053 are more potent thananthramycin against these ovarian tumour cell lines.

(b) Compounds of Formula II IC₅₀/μM UP No. A2780 A2780cisR CH1 CH1cisRSkov3 Anthramycin 0.155 0.16 0.062 0.05 0.16 UP2001 (80) 0.0000230.000024 0.00012 0.0006 0.0091 UP2004 (70) 0.029 0.2 0.017 0.082 0.35UP2023 (64) 0.49 1.45 0.37 0.43 16 UP2064 (74) 0.15 0.36 0.066 0.0840.39 UP2067 (172) 0.115 0.39 0.165 0.18 0.54 UP2100 (207) <0.05 0.066<0.05 <0.05 0.081Compound UP2100 (207) has the sructural; formula:

and was synthesised by the same route as compound 70.

UP2001 (80) exhibits cytotoxicity at picomolar/sub nanomolar levelsacross the ovarian tumour cell line panel. The potency of the moleculeis probably due to its cross-linking properties coupled with the effectof exo saturation. UP2001 is markedly more potent than UP2053.

The monomers UP2004 (70) and UP2100 (206) exhibit good activity againstthe ovarian tumour cell lines comparable to tjat for anthramycin. UP2023(64), which possesses a 7-iodo substituent is significantly less activethan UP2004 (70), which contains two alkoxy groups at the 7 and 8positions.

(c) Compounds of Formula III IC₅₀/μM Compound A2780 A2780cisR CH1CH1cisR Skov3 UP2020 (90) 10 7.2 1.7 2.8 1.6 UP2021 (130) >100 >100 5147 >100 UP2022 (143) 16.5 14 11 33 UP2024 (101) 1.4 1.8 1.45 1.25 2.35UP2025 (106) 0.064 0.155 0.082 0.11 1.7 UP2026 (136) 1.15 3.7 1.5 1.454.9 UP2027 (138) 0.56 1.55 1.35 1.15 1.7 UP2029 (140) 34.5 32 22.5 141.4 UP2066 (113) 11 12 3.8 7.4 15 UP2068 (96) 0.47 0.66 0.52 0.42 0.76UP2086 (120) 0.84 0.45 1.6 2.2 2.5UP2025 is the most potent monomer with two methoxy groups donatingelectrons to the A-ring, however some compounds with 3 electron donatinggroups appear to be less cytotoxic (eg. UP2020-2022 and UP2066).

The simple phenyl substituted PBD (UP2026, 136) shows micromolaractivity in the ovarian tumour cell lines. Introducing an electrondonating methoxy group into the phenyl substituent increasescytotoxicity (138) but the presence of an electron withdrawing nitrogroup reduces cytotoxic activity (140).

(d) Compounds of Formula IV IC₅₀/μM Compound A2780 A2780cisR CH1 CH1cisRSkov3 UP2005 (161) 1.5 4.3 1.4 1.85 5.4 UP2006 (163) 3.2 14.5 4.9 7.923.5 UP2007 (165) 1.55 4.9 1.5 3.0 5.8 UP2008 (167) 0.23 0.94 0.24 0.421.45 UP2088 (205) 11 8.5 12 16 14

1. A compound of the formula:

wherein p is
 3. 2. A method of treatment of lung, colon, CNS, ovarian orbreast carcinomas or melanomas of the human or animal body comprisingadministering to such a subject a therapeutically effective amount of acompound according to claim
 1. 3. A method of treatment of acisplatin-refractory ovarian carcinoma of the human or animal bodycomprising administering to such a subject a therapeutically effectiveamount of a compound according to claim
 1. 4. A method of inhibiting thegrowth of human ovarian cisplatin-refactory cells which method comprisestreating said cells with a compound according to claim
 1. 5. Apharmaceutical composition comprising a compound of formula:

wherein p is 3 and a pharmaceutically acceptable excipient, carrier,buffer or stabilizer.